EphA2 T-cell epitopes and uses therefor

ABSTRACT

EphA2 T-cell epitope are provided herein. The epitopes include peptides corresponding to specific fragments of human EphA2 protein containing one or more T-cell epitopes, and conservative derivatives thereof. The EphA2 T-cell epitopes are useful in an assay, such as an ELISPOT assay, that may be used to determine and/or quantify a patient&#39;s immune responsiveness to EphA2. The epitopes also are useful in methods of modulating a patient&#39;s immune reactivity to EphA2, which has substantial utility as a treatment for cancers that overexpress EphA2, such as renal cell carcinoma (RCC). The EphA2 epitopes also can be used to vaccinate a patient against EphA2, by in vivo or ex vivo methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/233,796, filed Sep. 23, 2005 now U.S. Pat. No. 7,297,337, which is acontinuation of U.S. application Ser. No. 10/897,711, filed on Jul. 22,2004 now abandoned and published on Mar. 3, 2005 as U.S. PatentApplication Publication No. 2005/0048550 A1, which claims the benefit ofU.S. Provisional Patent Application No. 60/491,046, filed Jul. 30, 2003,all of which are incorporated herein by reference in their entirety.

This work was supported by National Institutes of Health (NIH) grantNos. CA57840 and CA56937.

BACKGROUND

1. Field of the Invention

Eph2A T-cell epitopes are provided. The Eph2A T-cell epitopes are usefulin methods for diagnosing cancer, for quantifying EphA2-reactive T-cellsin a patient and in eliciting an immune response to EphA2 and modulatingthe immune system to recognize cancerous cells.

2. Description of the Related Art

The molecular identification of tumor antigens recognized by the immunesystem has paved the way for the development of new immunotherapeuticstrategies for the treatment of cancer. While many cytotoxic Tlymphocyte (CTL)-defined tumor-associated epitopes have been appliedclinically in cancer vaccinations (Coulie P G, et al. Proc Natl Acad SciUSA 98: 10290-1295, 2001; Yu J S, et al. Cancer Res 61: 842-847, 2001;Jager E, et al. Proc Natl Acad Sci USA 97: 12198-12203, 2000; and NestleF O, et al. Nat Med 4:328-332, 1998.), comparatively few classII-restricted epitopes recognized by CD4⁺ T cells have been identifiedand clinically-integrated to date (Topalian S L, et al. Proc Natl AcadSci USA 91; 9461-9465, 1994; Chaux P, et al. J Exp Med 189; 767-777,1999; Pieper R, et al. J Exp Med 189; 757-765, 1999; Wang R F, et al.Science 284; 1351-1354, 1999; Topalian S L, et al. J Exp Med 183;1965-1971, 1996; Jager E, et al. J Exp Med 191; 625-630, 2000; Zarour HM, et al. Cancer Res 60; 4946-4952, 2000; and Zarour H M, et al. ProcNatl Acad Sci USA 97; 400-405, 2000). Current paradigms suggest thatCD4⁺ T cells (at least Th1-type) play critical roles in the optimalinduction and maintenance of clinically beneficial tumor immunity(Pardoll D M, et al. Curr Opin Immunol 10; 588-594, 1998 and Toes R E,et al. J Exp Med 189; 753-756, 1999). Hence, CD4⁺ and CD8⁺ T cellepitopes derived from antigens that are unique to, or that areoverexpressed on tumor cells may provide effective vaccine components.

The Eph family of molecules constitutes the largest family of receptortyrosine kinases in the human genome. Eph kinases include two majorclasses (EphA and EphB), which are distinguished by their specificitiesfor the ligands ephrin-A and ephrin-B, respectively (Eph NomenclatureCommittee. Unified nomenclature for Eph family receptors and theirligands. The ephrins, Cell 90; 403-404, 1997). Largely known for theirrole in neuronal development, recent reports suggest that Eph receptorsplay a role in carcinogenesis. For example, EphA2 is overexpressed andfunctionally altered in a large number of different cancers, where itappears to promote the development of disseminated disease. In normalcells, EphA2 localizes to sites of cell-to-cell contact, where it mayplay a role as a negative regulator of cell growth. In contrast, EphA2is frequently overexpressed and often functionally dysregulated inadvanced cancers, where it contributes to many different aspects ofmalignant character. These changes in EphA2 have been observed in a widearray of solid tumors, including melanoma, prostate, breast and lungtumors. The highest degree of EphA2 expression among tumors is mostcommonly observed in metastatic lesions.

In the clinical setting, several findings suggest that T cell-mediatedimmunity provides a safeguard against the development and progression ofrenal cell carcinoma (RCC) and may effectively mediate the regression ofestablished lesions. RCC lesions are typically infiltrated with largenumbers of lymphocytes, though the benefits of leukocytic infiltrationupon beneficial clinical outcome remain unknown. While this may reflectvariance in the functional subsets of CD4⁺ and CD8⁺ T cells in theseinfiltrates, data addressing the prognostic benefit of Th1/Tc1-biasedimmunity versus Th2/Tc2-biased immunity in RCC patients has beenequivocal. A better understanding of the constitutive nature andspecificity of CD8⁺ and CD4⁺ T cell responses in RCC patients willlikely provide insights necessary to design, implement and monitor moreeffective treatment options.

SUMMARY

Provided herein are novel EphA2 T-cell epitopes and uses therefor,including diagnostic and prognostic methods, methods for eliciting animmune response to EphA2 and treatments for cancer. The epitopes areuseful in the detection and staging of RCC. It is demonstrated hereinthat high levels of EphA2 expression are observed in the setting ofrenal cell carcinoma (method of staging RCC) and that patients with RCCexhibit both CD8⁺ and CD4⁺ T cell responses to novel EphA2-derivedepitopes. Moreover, the reactivity of T cells against EphA2 is useful indistinguishing disease status and outcome and the EphA2 T-cell epitopesdescribed herein are useful in eliciting an immune response to EphA2, asa cancer therapy.

In one embodiment, an EphA2 T-cell epitope is provided comprising anEphA2 T-cell epitope. The EphA2 T-cell epitope may be a peptidecomprising an EphA2 T-cell epitope. In certain embodiments, the peptideconsists of from about 9 to about 35 amino acids, from about 9 to about25 amino acids or less than about 20 amino acids. The peptide can be aportion or fragment of native human EphA2 (SEQ ID NO: 2) and typicallycomprises at least about 9 contiguous amino acids of SEQ ID NO: 2 or aconservative derivative of a portion of SEQ ID NO: 2 in which one ormore amino acid residues are inserted into the peptide or one or moreamino acids of SEQ ID NO: 2 are deleted from the peptide or substitutedwith one or more different amino acid residues, so long as the bindingof the conservative derivative to an MHC molecule is substantially equalto or enhanced as compared to binding of EphA2 or a fragment thereof tothe MHC molecule.

The EphA2 T-cell epitope can be a modified peptide comprising one ormore of N-terminal modifications, C-terminal modifications, internalmodifications or non-standard residues, for example and withoutlimitation, a solubilizing group; a hydrophobic group; a lipid group; ahydrophilic group; a tag; a fluorescent tag; a polypeptide tag; atransmembrane signal sequence or a portion thereof; an amino acidenantiomer and one of an acetyl, benzyloxycarbonyl, biotin, cinnamoyl,dabcyl, dabsyl, dansyl, dinitrophenyl, cyanine, fluorescein, fmoc,formyl, lissamine rhodamine, myristoyl, n-methyl, palmitoyl, steroyl,7-methoxycoumarin acetic acid, biotin, dabcyl, dabsyl, dansyl,disulphide, acetamidomethyl, aminohexanoic acid, aminoisobutyric acid,beta alanine, cyclohexylalanine, d-cyclohexylalanine, e-acetyl lysine,gamma aminobutyric acid, hydroxyproline, nitro-arginine,nitro-phenylalanine, nitro-tyrosine, norleucine, norvaline,octahydroindole carboxylate, ornithine, penicillamine, phenylglycine,phosphoserine, phosphothreonine, phosphotyrosine, L-malonyltyrosine,pyroglutamate, tetrahydroisoquinoline, amide, N-substituted glycine;non-amino acyl and N-acetylglycine group. In certain embodiments, theEphA2 T-cell epitope is a peptoid or a peptidomimetic comprising anEphA2 T-cell epitope.

In certain embodiments, the EphA2 T-cell epitope comprises a T-cellepitope contained in one or more of the following EphA2 epitopesequences: TLADFDPRV (SEQ ID NO: 2, residues 883-891); VLLLVLAGV (SEQ IDNO: 2, residues 546-554); VLAGVGFFI (SEQ ID NO: 2, residues 550-558);IMNDMPIYM (SEQ ID NO: 2, residues 58-66); SLLGLKDQV (SEQ ID NO: 2,residues 961-969); WLVPIGQCL (SEQ ID NO: 2, residues 253-261); LLWGCALAA(SEQ ID NO: 2, residues 12-20); GLTRTSVTV (SEQ ID NO: 2, residues391-399); NLYYAESDL (SEQ ID NO: 2, residues 120-128); KLNVEERSV (SEQ IDNO: 2, residues 162-170); IMGQFSHHN (SEQ ID NO: 2, residues 666-674);YSVCNVMSG (SEQ ID NO: 2, residues 67-75); MQNIMNDMP (SEQ ID NO: 2,residues 55-63) and a sequence presented in one or more of FIGS. 5-17.

As a non-limiting example, the EphA2 T-cell epitope can comprise apeptide, or a modified version thereof, comprising one or more of thefollowing amino acid sequences: TLADFDPRV (SEQ ID NO: 2, residues883-891); VLLLVLAGV (SEQ ID NO: 2, residues 546-554); VLAGVGFFI (SEQ IDNO: 2, residues 550-558); IMNDMPIYM (SEQ ID NO: 2, residues 58-66);SLLGLKDQV (SEQ ID NO: 2, residues 961-969); WLVPIGQCL (SEQ ID NO: 2,residues 253-261); LLWGCALAA (SEQ ID NO: 2, residues 12-20); GLTRTSVTV(SEQ ID NO: 2, residues 391-399); NLYYAESDL (SEQ ID NO: 2, residues120-128); KLNVEERSV (SEQ ID NO: 2, residues 162-170); IMGQFSHHN (SEQ IDNO: 2, residues 666-674); YSVCNVMSG (SEQ ID NO: 2, residues 67-75);MQNIMNDMP (SEQ ID NO: 2, residues 55-63) and a sequence presented in oneor more of FIGS. 5-17, or a conservative derivative thereof. In oneembodiment, the EphA2 T-cell epitope comprises two or more EphA2 T-cellepitopes separated by a spacer.

A composition is provided that comprises one or more EphA2 T-cellepitope as described above and a pharmaceutically acceptable carrier.

In another embodiment, a method of monitoring the number and/or statusof EphA2-reactive T-cells in a patient is provided, the method comprisesdetermining the patient's immune reactivity to a compound or compositioncontaining an EphA2 T-cell epitope containing one or more EphA2 T-cellepitopes, as described above. In one embodiment, the method comprisesdetermining the patient's immune reactivity to a compound or compositioncontaining one or more EphA2 T-cell epitopes using an ELISPOT assay. TheELISPOT assay may detect a CD8⁺ response to an MHC class Iprotein-presented EphA2 epitope or a conservative derivative thereof.The MHC class I protein can be an HLA-A2 protein. The ELISPOT assay alsomay detect a CD4⁺ response to an MHC class II protein-presented EphA2epitope or a conservative derivative thereof. The MHC class II proteincan be an HLA-DR4 protein.

In a further embodiment, a method is provided for inhibiting growth in apatient of a cancer in which EphA2 is overexpressed, comprisingadministering to the patient an amount of an EphA2 T-cell epitope asdescribed above, effective to elicit an immune response to EphA2 in thepatient. In one embodiment, the method comprises contacting anantigen-presenting cell of a patient with the EphA2 T-cell epitope. Inanother embodiment, the method is an ex vivo method comprising:isolating cells comprising an antigen-presenting cell from the patient;contacting the antigen-presenting cell with the EphA2 T-cell epitope;and reintroducing the EphA2 T-cell epitope-contacted antigen-presentingcell into the patient. The method may further comprise administering tothe patient an EphA2 ligand or agonist thereof, such as, withoutlimitation, a binding reagent capable of binding to EphA2; and ephrinA1or an agonist thereof.

Also provided is an isolated nucleic acid comprising from 5′ to 3′ andoperably linked, a promoter, a coding sequence, other than a full lengthEphA2 coding sequence, encoding a peptide comprising one or more EphA2T-cell epitopes and a polyadenylation signal. The nucleic acid is usefulin preparing the EphA2 T-cell epitope by recombinant methods and/or bytransfer of the nucleic acid into a patient's cells, either ex vivo orin vivo, to produce the EphA2 T-cell epitope in vivo.

In another embodiment, a method is provided comprising contacting atumor cell that expresses EphA2 on its surface with an EphA2 ligand oragonist thereof comprising one of: a binding reagent capable of bindingto EphA2; and ephrinA1 or an agonist thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 provide the amino acid (SEQ ID NO: 2) and nucleic acid(SEQ ID NO: 1) sequences of human EphA2 (GenBank Accession Nos. AAH37166and BC037166 (also NM_(—)004431), respectively).

FIGS. 3 and 4 provide non limiting lists of human MHC Class II and ClassI alleles, respectively.

FIGS. 5-8 provide in silico predicted MHC Class I binding peptideswithin the EphA2 amino acid sequence for the Class I alleles HLA-A1,HLA-A3, HLA-B7 and HLA-B44, respectively (portions of SEQ ID NO: 2, asindicated). In FIGS. 5-8, the “Score” refers to an estimate of the halftime of dissociation (T_(1/2)) of a molecule containing the listedsequence.

FIGS. 9-17 provide in silico predicted MHC Class II binding peptideswithin the EphA2 amino acid sequence for the Class II allelesHLA-DRβ1*0101, HLA-DRβ1*0301, HLA-DRβ1*0701, HLA-DRβ1*0801,HLA-DRβ1*1101, HLA-DRβ1*1301, HLA-DRβ1*1501 and HLA-DRβ5*0101,respectively (portions of SEQ ID NO: 2, as indicated). In FIGS. 9-17,the “Score” refers to a comparison of binding to a theoretical positivecontrol generated by the software used to identify the peptides.

FIG. 18 is a Western blot showing analysis of lysates generated from theindicated RCC cell lines.

FIG. 19 are photomicrographs showing expression of EphA2 in RCC celllines.

FIG. 20 provides graphs showing IFN-γ ELISPOT (enzyme-linked immunospot)analysis of RCC patient CD8⁺ T cell responses to EphA2-derived epitopesversus disease status.

FIG. 21 provides graphs showing IFN-γ ELISPOT analysis of RCC patientCD8⁺ T cell responses to EphA2-derived epitopes versus disease stage.

FIG. 22 provides graphs showing observed changes in peripheral bloodCD8+ T cell responses to EphA2 epitopes pre-versus post-surgery in 4HLA-A2⁺ patients with RCC.

FIG. 23 provides graphs showing disease-stage skewing of functional CD4⁺T cell responses to EphA2 Th epitopes in HLA-DR4⁺ RCC patients withactive disease.

FIG. 24 provides graphs showing therapy-associated enhancement ofTh1-type, and reduction in Th2-type, CD4⁺ T cell responses to EphA2 Thepitopes in an HLA-A2⁺/DR4⁺ patient with Stage I RCC.

FIG. 25 provides graphs showing suppressor CD4⁺ T cell responses toEphA2 Th epitopes in HLA-DR4⁺ patients with advanced Stage IV RCC.

FIG. 26 is a Western blot showing that EphA2 agonists induce thephosphorylation of EphA2.

FIG. 27 shows that EphA2 agonists induce the degradation of EphA2.

FIGS. 28A and 28B show that EphA2 agonists-induced degradation isinhibited by MG132, but not by chloroquine.

FIG. 29 is a graph showing that EphA2 agonists sensitize the RCC cellline SLR24 to recognition by anti-EphA2 CD8⁺ T cell clone CL.142.

FIGS. 30A and 30B demonstrate “agonistic” triggering of tumor cell EphA2in situ enhances the therapeutic efficacy of adoptively transferredanti-EphA2 specific CD8⁺ T cells.

DETAILED DESCRIPTION

Provided herein are EphA2 T-cell epitopes. The epitopes are compoundscontaining one or more T-cell epitopes of EphA2 and typically arepeptides corresponding to portions of the EphA2 amino acid sequence(FIG. 1, SEQ ID NO: 2). Also provided are methods for making theepitopes and recombinant systems for production of the epitopes. TheEphA2 T-cell epitopes are useful in methods for determining a patient'simmune status, or immune reactivity to EphA2 by quantifying the numberof EphA2-reactive T-cells in the patient. The epitopes also are usefulin modulating a patient's immune responsiveness to EphA2 as a cancertreatment.

As used herein, the term “agonist” is a ligand that is capable ofcombining with (binding) a receptor on a cell and initiating a reactionor activity that mimics the activity of a natural ligand, which, in thecontext of the present disclosure is native EphA2 as shown in FIG. 1.The term “epitope” refers to a physical structure that contains and/ordefines an antigenic determinant. “Peptide agonists” are peptides,peptide derivatives or peptide analogs that mimic a naturally-occurringligand, which, in the context of the present disclosure, is an EphA2ligand.

The term “binding reagent” and like terms, refers to any compound,composition or molecule capable of specifically or substantiallyspecifically (that is with limited cross-reactivity) binding anothercompound or molecule, which, in the case of immune-recognition containsan epitope. Typically, the binding reagents are antibodies, preferablymonoclonal antibodies, or derivatives or analogs thereof, includingwithout limitation: Fv fragments; single chain Fv (scFv) fragments; Fab′fragments; F(ab′)2 fragments; humanized antibodies and antibodyfragments; camelized antibodies and antibody fragments; and multivalentversions of the foregoing. Multivalent binding reagents also may beused, as appropriate, including without limitation: monospecific orbispecific antibodies, such as disulfide stabilized Fv fragments, scFvtandems ((scFv)₂ fragments), diabodies, tribodies or tetrabodies, whichtypically are covalently linked or otherwise stabilized (i.e., leucinezipper or helix stabilized) scFv fragments. “Binding reagents” alsoinclude aptamers, as are described in the art.

Methods of making antigen-specific binding reagents, includingantibodies and their derivatives and analogs and aptamers, arewell-known in the art. Polyclonal antibodies can be generated byimmunization of an animal. Monoclonal antibodies can be preparedaccording to standard (hybridoma) methodology. Antibody derivatives andanalogs, including humanized antibodies can be prepared recombinantly byisolating a DNA fragment from DNA encoding a monoclonal antibody andsubcloning the appropriate V regions into an appropriate expressionvector according to standard methods. Phage display and aptamertechnology is described in the literature and permit in vitro clonalamplification of antigen-specific binding reagents with very affinitylow cross-reactivity. Phage display reagents and systems are availablecommercially, and include the Recombinant Phage Antibody System (RPAS),commercially available from Amersham Pharmacia Biotech, Inc. ofPiscataway, N.J. and the pSKAN Phagemid Display System, commerciallyavailable from MoBiTec, LLC of Marco Island, Fla. Aptamer technology isdescribed for example and without limitation in U.S. Pat. Nos.5,270,163, 5,475,096, 5,840,867 and 6,544,776.

A “gene” is an operative genetic determinant in its broadest sense. Agene includes an “expressed sequence” that encodes a protein or istranscribed into a functional RNA product, for example an open readingframe (ORF). A typical gene includes an expressed sequence, along withoperably linked regulatory sequences, including, but not limited to,promoters, enhancers, transcription factor binding sequences, operatorsand terminators (for example poly(A) sequences). Promoters can be, forexample and without limitation, constitutive or semi-constitutive (forexample, CMV and RSV promoters), tissue-specific promoters (for example,a muscle creatinine kinase (MCK) promoter) or induceable (for exampleand without limitation tetracycline-regulatable systems, such as the BDTet-On™. and BD Tet-Off™. Gene Expression Systems, commerciallyavailable form BD Biosciences Clontech of Palo Alto, Calif.). Twosequences are considered to be “operably linked” if they are arranged incis to act in an expected manner in relationship to each other. In agene, regulatory sequences are operably linked in a manner sufficient tocause correct and/or desired transcription of the expressed sequence ina cell. The terms “expression” or “gene expression,” and like words andphrases, mean the overall process by which the information encoded in anucleic acid, typically a gene, is converted into a ribonucleic acidand/or a protein, or a post-translationally modified version thereof,and/or an observable phenotype.

As used herein, a “nucleic acid” may be, without limitation, anypolynucleotide or polydeoxynucleotide. Without limitation, a nucleicacid may be single-stranded or double stranded. In context of the humanEphA2 peptide and nucleotide sequences disclosed herein (FIG. 1, SEQ IDNO: 2 and FIG. 2, SEQ ID NO: 1, respectively), reference is made toconservative derivatives. A “conservative derivative” is a nucleic acidor a peptide containing conservative substitutions, which include, inthe case of a nucleic acid, substitutions with nucleotide bases thataccount for codon degeneracy, for example and without limitation inreference to the Ala codon, the substitution of GCC or GCG for GCA, or,in the case of both nucleic acids and peptides, that representconservative amino acid substitutions, including but not limited to theconservative substitution groups: Ser and Thr; Leu, Ile and Val; Glu andAsp; and Gln and Asn. Conservative substitutions also may be determinedby other methods, such as, without limitation, those used by the BLAST(Basic Local Alignment Search Tool) algorithm, such as a BLOSUMSubstitution Scoring Matrix, such as the BLOSUM 62 matrix. Importantly,a conservative derivative substantially retains the function of thenative nucleic acid or peptide to which the conservative derivativecorresponds. In the context of an EphA2 T-cell epitope, a conservativederivative, as with all “derivatives,” substantially retains the abilityto stimulate an appropriate immune response to EphA2 in the assaysdescribed herein.

The similarity between two nucleic acid or protein sequences may bedetermined by a variety of methods. For example, the similarities may bedetermined in silico by an algorithm, for example a BLAST algorithm,which is the reference standard used herein. The similarity between twonucleic acid sequences also may be determined by specific hybridization,which means that a nucleic acid will hybridize specifically in a genometo a reference nucleic acid (namely, the EphA2 sequence provided hereinor portions thereof). The hybridization conditions for achievingspecificity naturally will differ, depending on such factors including,without limitation, the length of sequence overlap of the respectivenucleic acids, its (melting temperature) Tm, the specific genome and theassay conditions.

“Derivatives” also include chemically modified nucleic acids or peptidescorresponding to portions of the EphA nucleotide or amino acid sequenceand conservative derivatives thereof. The nucleic acids or peptides, orconservative derivatives thereof may be derivatized to contain chemicalgroups that, for example: modify the solubility of the nucleic acid orpeptide, for example by the addition of a PEG group; permit affinitypurification of the peptide or nucleic acid, for example by the additionof a biotin or poly(his) tag;) or permit detection of the compound, forexample by conjugation with a fluorochrome, such as fluoresceinisothiocyanate, Cy3 or Cy5 or a radionuclide-containing or caging groupfor in vitro or in vivo detection and location of the nucleic acid,peptide or derivative thereof. These examples of modified nucleic acidsand peptides, and used therefor, are only limited examples of the largevariety of useful modifications of nucleic acids and peptides that areknown in the art. A more complete, but non exhaustive list of suchmodifications include one or more of N-terminal modifications,C-terminal modifications, internal modifications or non-standardresidues for example, and without limitation the following groups and/orresidues: a solubilizing group (such as, without limitation apolyethylene glycol (PEG) group), a hydrophobic group, a lipid group, ahydrophilic group, a tag (such as, without limitation: a fluorescent tag(such as fluorescein (e.g., FITC) or a cyanine dye (e.g., Cy3 or Cy5))or a polypeptide tag (e.g., poly-histidine, for affinity purification,for example)), a transmembrane signal sequence or a portion thereof, anamino acid enantiomer, acetyl, benzyloxycarbonyl, biotin, cinnamoyl,dabcyl, dabsyl, dansyl, dinitrophenyl, cyanine, fluorescein, fmoc,formyl, lissamine, rhodamine, myristoyl, n-methyl, palmitoyl, steroyl,7-methoxycoumarin acetic acid, biotin, dabcyl, dabsyl, dansyl,disulphide, acetamidomethyl, aminohexanoic acid, aminoisobutyric acid,beta alanine, cyclohexylalanine, d-cyclohexylalanine, e-acetyl lysine,gamma aminobutyric acid, hydroxyproline, nitro-arginine,nitro-phenylalanine, nitro-tyrosine, norleucine, norvaline,octahydroindole carboxylate, ornithine, penicillamine, phenylglycine,phosphoserine, phosphothreonine, phosphotyrosine, L-malonyltyrosine,pyroglutamate, tetrahydroisoquinoline, amide, N-substituted glycinesand/or non-amino acyl groups (peptoids), N-acetylglycine.

“Derivatives” also include peptide analogs, which are peptidescontaining one or more modified bases and/or a modified peptide backbone(a typical or normal peptide backbone having the structure: . . .—NH—CR—CO—NH—CR—CO—NH—CR—CO— . . . ). Peptide analogs include“peptidomimetics”, which are compounds containing one or morenon-peptidic structural elements that are capable of mimicking orantagonizing the biological action(s) of a natural parent peptide. Apeptidomimetic does not have classical peptide characteristics such asenzymatically scissile peptide bonds. A common peptidomimetic is a“peptoid”, which is a polymer that that includes one or moreN-substituted amino acid residues, such as N-substituted glycine.Non-limiting examples of peptoids, peptoid synthesis methods, uses forpeptoids and methods of using peptoids are provided in Simon, R. et al.(1992), Proc. Natl. Acad. Sci. USA, 89:9367-9371; Murphy, J. E. et al.,(1998) Proc. Natl. Acad. Sci. USA, 95:1517-1522 and in U.S. Pat. Nos.5,811,387, 5,877,278, 5,965,695 and 6,075,121, which are incorporatedherein by reference for their teachings of peptoid structures, peptoidsynthesis methods, uses for peptoids and methods of using peptoids.

In the examples, certain T-cell epitopes of EphA2 are described andanalyzed for their ability to elicit an EphA2-specific immune response.Those epitopes were identified in silico in the context of the MHC ClassII allele HLA-DR.beta.1*0401 (DR4) or the Class I allele HLA-A0201(HLA-A2). HLA-DR.beta.1*0401 and HLA-A0201 are two alleles among many.Non-limiting examples of other Class II HLA-DR alleles are shown in FIG.3, including HLA-DR1, HLA-DR3, HLA-DR4, HLA-DR7, HLA-DR8, HLA-DR9,HLA-DR11, HLA-DR12, HLA-DR13, HLA-DR14 and HLA-DR15 alleles(www-dot-anthonynolan-dot-org-dot-uk-slashHIG-slash-lists-slash-class2list-dot-html).More common Class II alleles include, without limitation: HLA-DR2,HLA-DR3, HLA-DR4 and HLA-DR5. Southwood et al., 1998, Honeyman et al.,1998 and De Groot et al., Vaccine (2001) 19:4385-4395 describealgorithms, consensus sequences and other methods for identifyingMHC-binding sequences in connection with a variety of HLA-DR.beta.1alleles. By applying the algorithms described in those references, orother algorithms that search for consensus MHC-II binding sequences(such as the ProPred software/algorithms referenced herein), other EphA2MHC-II-containing fragments can be identified that are specific toalleles other than HLA-DR.beta.1*0401 or HLA-A0201. Once the consensusMHC II binding sequences are identified, the algorithm described belowfor use in identifying proteasomal cleavage products, or any likealgorithms, can be used to select candidate testing for screening inELISPOT assays and ELISA assays as described herein, or like assays, forimmunostimulatory activity in EphA2-reactive PBLs from a patient withthe same MHC haplotype.

As with the Class II alleles described above, a large number of MHCclass I alleles other than HLA-A0201 also have been identified and EphA2T-cell epitopes specific to those alleles can be determined in a likemanner. Non-limiting examples of MHC class I HLA-A or HLA-B alleles areprovided in FIG. 4(www-dot-anthonynolan-dot-org-dot-uk-slash-HIG-slash-lists-slash-classIlist-dot-html).More common alleles include, without limitation: HLA-A1, HLA-A2, HLA-A3,HLA-B7 and HLA-B44.

As indicated above, the consensus binding sequences have been resolvedfor many of the MHC Class I and II alleles provided in FIGS. 3 and 4.FIGS. 5-8 provide in silico predicted MHC Class I binding peptideswithin the EphA2 amino acid sequence for the Class I alleles HLA-A1,HLA-A3, HLA-B7 and HLA-B44, respectively. FIGS. 9-17 provide in silicopredicted MHC Class II binding peptides within the EphA2 amino acidsequence for the Class II alleles HLA-DRβ1*0101, HLA-DRβ1*0301,HLA-DRβ1*0401, HLA-DRβ1*0701, HLA-DRβ1*0801, HLA-DRβ1*1101,HLA-DRβ1*1301, HLA-DRβ1*1501 and HLA-DRβ5*0101, respectively.

Although the available software useful in identifying MHC-consensusbinding regions contains consensus sequences for many MHC Class I andClass II alleles (including, without limitation, 39 Class I alleles,including HLA-A1, HLA-A24 and HLA-B7 alleles, available for searching inthe NIH BIMAS “HLA Peptide Binding Predictions” software (for example,athttp-colon-slash-slash-bimas-dot-dcrt-dot-nih-dot-gov-slash-molbio-slash-hla_bind-slash)and 51 Class II alleles, including 49 HLA-DR.beta.1 alleles and 2HLA-DR.beta.5 alleles, Singh et al., ProPred: prediction of HLA-DRbinding sites Bioinformatics (2001) December; 17(12):1236-7; both ofwhich were utilized to identify the putative EphA2 Class I and Class IIT-cell epitopes identified in one or more of FIGS. 5-17), methods foridentifying consensus binding sequences are well-described in theliterature. For example, Luckey et al., 2001 describes methods foridentifying Class I binding sequences—briefly by the steps ofacid-treating cells to elute Class I molecules, affinity purifying thevarious alleles, eluting bound peptides form the affinity-purified HLAmolecules and sequencing the eluted peptides. Methods for identifyingconsensus binding sequences for each allele, and the recognition thatmany alleles can bind the same or very similar peptide sequencerepertoires (HLA supertypes) is discussed in Southwood et al., 1998.Nevertheless, the overall goal is to identify specific EphA2 T-cellepitopes, which can be accomplished by eluting processed EphA2 peptidefragments from any MHC molecule purified from a cell, such as an APC(antigen presenting cell), for any MHC allele, and sequencing the elutedpeptides, all according to well-established methods. This completelyavoids the in silico step.

The EphA2 T-cell epitopes described herein, can be used in ELISPOT, orlike assays, to screen patients for their immune reactivity to EphA2,and can be used to stimulate a patient's immune response to EphA2. Inthis manner an immunogenic composition or cocktail can be prepared for agiven patient, depending on that patient's HLA-DR haplotype. As alsomentioned herein, an immune response to sub-dominant EphA2 T-cellepitopes can be elicited in a patient, which could overwhelm thepatient's tolerance to one or more dominant epitopes.

The EphA2 T-cell epitope compounds are useful in an assay to establish apatient's existing immunity to EphA2. As described herein, a populationof a patient's PBLs may be stimulated with a compound containing one ormore EphA2 T-cell epitopes, as described herein. The one or more EphA2T-cell epitopes are selected to match the patient's MHC haplotype.Hence, if the patient has the HLA-DRβ1*0701 allele, a compoundcontaining one or more EphA2 HLA-DRβ1*0701-binding peptides is selected.Once stimulated with the compound for a sufficient period of time(typically 6 hours to 48 hours), the PBL population is tested forstimulation by the antigen. This testing can be performed by a varietyof methods, such as by ELISPOT to determine IFN-γ or IL-5 production, orELISA to determine TGF-β or IL-10 production, purportedly indicative ofantigen-specific suppression.

A number of assays are used to detect antigen-specific immune responses(see, Keilholz, U. et al., “Immunologic Monitoring of Cancer VaccineTherapy: Results of a Workshop Sponsored by the Society for BiologicalTherapy,” J. Immunother., (2002) 25(2):97-138). The ELISPOT assaydescribed herein is quite sensitive and accurate. Other assays that arepromising substitutes include, but are not limited to: 1) Cytokine FlowCytometry (CFC), in which cytokine production is detectedintracellularly in a cell population and which only requires about a sixhour stimulation period; 2) MHC-peptide tetramer analysis in whichisolated MHC-peptide tetramers are used to stimulate an antigen-specificresponse in PBL and bound cells can be counted by flow cytometry; and 3)Quantitative Reverse Transcription Polymerase Chain Reaction (QRT-PCR)assays in which the expression of one or more gene target, such ascytokines, can be monitored, permitting rapid quantitation of expressionlevels from a small sample of cells. Each of these assays are describedin further detail in Keilholz et al., 2002 and in the literature. Any ofthe described assays may be used alone, or in combination with others todetermine if a patient's PBL are capable of producing a suitableantigen-specific response. Of note, the compounds containing the EphA2T-cell epitopes described herein are useful in the ELISPOT, CFC andtetramers assays described above, but not in the QRT-PCR assay, whichrequires design of suitable PCR primer and probe sets according toestablished methods.

Image analysis-assisted cytokine ELISPOT assay is a sensitive method fordirect ex vivo monitoring of antigen-specific CD4⁺ or CD8⁺ T cells. Theprocedure measures both the frequency and cytokine signatures ofantigen-specific T cells in freshly isolated cellular material. Theassay determines various parameters of T cell immunity such as theclonal size (magnitude) and the Th1/Th2 effector class of the T cellpool. ELISPOT is a superior method through which the actual secretoryprocesses of individual pharmacologically unmanipulated cells can bestudied. The technology is non destructive and the lymphocytes can bepreserved for further analysis. Under the ELISPOT technique, cytokinerelease can be detected at the single cell level, allowing for thedetermination of cytokine-producing cell frequencies. The ELISPOT assayuses plates coated with an antibody, typically an anti-cytokineantibody. In the Examples below, the plates are coated with IL-5 (Th2cytokine profile) and IFN-γ (Th1 cytokine profile). The ELISPOT assayincludes the steps of incubating cytokine producing cells, such as PBLs,in the antibody-coated plates in the presence of an antigen. The cellsare washed away, leaving just the antibodies, some of which will bebound to its cytokine ligand. A standard “sandwich assay” is thenperformed in which tagged or labeled anti-cytokine antibody is bound tothe previously bound cytokine and is detected by standard methods, suchas by a standard biotin-avidin-HRP (horseradish peroxidase) method.Bound cytokine is therefore represented on the plate as a spot at thesite of the complex. The colored spots are then counted and their sizeanalyzed either visually or more commonly by computer analysis,providing data that is then used to calculate the cytokine secretionfrequency.

Both ELISPOT assays and ELISAs are examples of sandwich assays. The term“sandwich assay” refers to an immunoassay where the antigen issandwiched between two binding reagents, which are typically antibodies.The first binding reagent/antibody being attached to a surface and thesecond binding reagent/antibody comprising a detectable group. Examplesof detectable groups include, for example and without limitation:fluorochromes, enzymes, epitopes for binding a second binding reagent(for example, when the second binding reagent/antibody is a mouseantibody, which is detected by a fluorescently-labeled anti-mouseantibody), for example an antigen or a member of a binding pair, such asbiotin. The surface may be a planar surface, such as in the case of anELISPOT assay or a typical grid-type array, as described herein, or anon-planar surface, as with coated bead array technologies, where each“species” of bead is labeled with, for example, a fluorochrome (such asLuminex technology, as described in U.S. Pat. Nos. 6,599,331, 6,592,822and 6,268,222), or a quantum dot (for example, as described in U.S. Pat.No. 6,306,610).

The epitopes described herein are compounds that contain one or moreEphA2 T-cell epitopes. The epitope can be, for example, with respect toT-cell epitopes defined by their binding to HLA-A2 and DR4, 1) peptideshaving one of the amino acid sequences: TLADFDPRV (SEQ ID NO: 2,residues 883-891); VLLLVLAGV (SEQ ID NO: 2, residues 546-554); VLAGVGFFI(SEQ ID NO: 2, residues 550-558); IMNDMPIYM (SEQ ID NO: 2, residues58-66); SLLGLKDQV (SEQ ID NO: 2, residues 961-969); WLVPIGQCL (SEQ IDNO: 2, residues 253-261); LLWGCALAA (SEQ ID NO: 2, residues 12-20);GLTRTSVTV (SEQ ID NO: 2, residues 391-399); NLYYAESDL (SEQ ID NO: 2,residues 120-128); KLNVEERSV (SEQ ID NO: 2, residues 162-170); IMGQFSHHN(SEQ ID NO: 2, residues 666-674); YSVCNVMSG (SEQ ID NO: 2, residues67-75); MQNIMNDMP (SEQ ID NO: 2, residues 55-63); or a sequence listedin one or more of FIGS. 5-17 or longer peptides containing thosesequences; 2) peptides containing derivatives or conservativederivatives of those peptide sequences, in which one or more amino acidsare deleted or are substituted with one or more different amino acids,the derivatives or conservative derivatives containing the T-cellepitopes defined by the peptide sequences listed above, 3) peptides,including fragments of EphA2, containing 2 or more of those peptidesequences or derivatives thereof, peptides containing the T-cell epitopedefined by those peptide sequences, or peptide analogs or othercompounds containing one or more of the T-cell epitopes defined by thosepeptide sequences.

In one embodiment, the epitope is a single peptide containing two ormore of the amino acid sequences containing the T-cell epitope of:TLADFDPRV (SEQ ID NO: 2, residues 883-891); VLLLVLAGV (SEQ ID NO: 2,residues 546-554); VLAGVGFFI (SEQ ID NO: 2, residues 550-558); IMNDMPIYM(SEQ ID NO: 2, residues 58-66); SLLGLKDQV (SEQ ID NO: 2, residues961-969); WLVPIGQCL (SEQ ID NO: 2, residues 253-261); LLWGCALAA (SEQ IDNO: 2, residues 12-20); GLTRTSVTV (SEQ ID NO: 2, residues 391-399);NLYYAESDL (SEQ ID NO: 2, residues 120-128); KLNVEERSV (SEQ ID NO: 2,residues 162-170); IMGQFSHHN (SEQ ID NO: 2, residues 666-674); YSVCNVMSG(SEQ ID NO: 2, residues 67-75); MQNIMNDMP (SEQ ID NO: 2, residues 55-63)or a sequence listed in one or more of FIGS. 5-17. Each sequence isseparated from the other by a peptide spacer that may be of any length,but typically ranges from 0 to 10 amino acids in length. In Velders etal. J. Immunol. (2001) 166:5366-5373, an AAY trimer spacer greatlyimproved the efficacy of an a Human Papilloma Virus (HPV16) multivalentepitope string vaccine. The peptide can be engineered to ensure thatprotease cleavage sites are located between the epitope sequences toensure proper processing of the peptide. This “string of beads”configuration can contain any number and combination of epitopes. DeGroot et al. 2001, Velders et al. 2001 and Ling-Ling et al. J. Virol.(1997) 71:2292-2302 describe peptides having this configuration, methodsfor making and optimizing such constructs, methods for identifyingcandidate epitopes and recombinant systems useful in making suchvaccines. The benefit of using a “string of beads” approach is thatsubdominant epitopes, or multiple copies of the same epitope may beincluded in a single peptide, thereby eliciting an immune response to anepitope or epitopes to which immunity is not normally elicited in aresponse to the native (i.e. EphA2) peptide. The concept of elicitingimmune responses to such cryptic or subdominant epitopes is called“epitope spreading” and can lead to a more robust immune response than atypical immune response to native peptides.

One or more epitopes also may be combined in a chimeric peptide with asecond amino acid sequence that has a desired functionality. Thefunctionality can be immunogenic in nature, permitting affinitypurification of the peptide. A protease cleavage site can be includedbetween the EphA2 T-cell epitope peptide and the immunogenic portion.The functionality also can facilitate the delivery of the EphA2 T-cellepitope peptide, by including amino acid sequences that facilitatedelivery of the peptide. One example of this is to include a portion oflactadherin or other protein to facilitate presentation of the peptideto dendritic cells in membrane vesicles or nanoparticles, such asexosomes. Methods for modifying and expressing chimeric peptides forincorporation into membrane vesicles are described in InternationalPatent Publication No. WO 03/016522.

The epitope, in any form described above, can be administered by anyuseful route to vaccinate or otherwise elicit an immune response in apatient. In one embodiment, the epitope is injected into the patient,optionally with an adjuvant, such as Freund's Incomplete Adjuvant,Freund's Complete Adjuvant, or as an exosome, as described above. Theepitope can be delivered in a variety of compositions which include theepitope and any desirable, pharmaceutically acceptable carrier.“Carrier” includes as a class, without limitation, any compound orcomposition useful in facilitating storage, stability, administrationand/or delivery of the active ingredients described herein, including,without limitation, suitable vehicles, solvents, diluents, excipients,pH modifiers, buffers, salts, colorants, flavorings, rheology odifiers,lubricants, coatings, fillers, antifoaming agents, erodeable polymers,hydrogels, surfactants, emulsifiers, adjuvants, preservatives,phospholipids, fatty acids, mono-, di- and tri-glycerides andderivatives thereof, waxes, oil and water, as are broadly known in thepharmaceutical arts. So long as the epitope is delivered to lymphoidcells, the route is immaterial. Atypical route of administration isintramuscular injection. The epitope can be administered once ormultiple times over a desired time period to elicit a desired immuneresponse. Suitable intervals for administering multiple doses typicallyrange from once a week to once a year, but typically ranges from onceevery seven to 90 days, and more typically, once every seven to 30 days.Optimal administration intervals may be gauged by a patient's immuneresponse, and the severity of the patient's condition. The amount of theepitope administered also may vary greatly, depending, among otherparameters, upon the structure of the epitope, the route of delivery andthe patient's health status. In any case, the amount of epitopeadministered at any given time to elicit an immune response to theepitope is an amount effective to do so. Similarly, the number of timesthe epitope is administered and the interval for administering multipledoses is a number and interval effective to elicit an immune response tothe epitope.

The epitope also may be delivered to a patient by liposomes. Liposomescan be directed to the site of lymphoid cells, where the liposomes thendeliver the selected epitope composition. Liposomes for use are formedfrom typical vesicle-forming lipids, which include neutral andnegatively charged phospholipids and a 5-sterol, such as cholesterol.The selection of lipids is generally guided by consideration of, forexample, liposome size, acid lability and stability of the liposomes inthe bloodstream. A variety of methods are available for preparingliposomes, as described in Szoka et al., Ann. Rev. Biophys. Bioeng.9:467 (1980) and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028 and5,019,369.

The epitopes also may be delivered in the presence of heat shockproteins. Heat shock proteins act as molecular chaperones in the cells,ensuring proper folding of nascent peptides into proteins and shuttlingnew and old proteins inside the cell. Heat shock proteins are believedto play a role in the presentation of peptides on the cell surface tohelp the immune system recognize diseased cells. U.S. Pat. Nos.5,935,576, 6,007,821 and 6,017,540 describe such uses for heat shockproteins, methods of making heat shock protein complexes and treatmentmethods.

An effective immune response also can be elicited by ex vivo methods. insuch methods, antigen is presented to PBL populations of antigenpresenting cells (often referred to as pulsing APCs), such as dendriticcells, obtained from a patient in vitro, and the immune-stimulated cellsare delivered back to the patient. This method ensures that the epitopesare delivered to the APCs and avoids both any potential toxicity to thepatient of the peptide and typically requires lesser amounts of epitope.Methods for isolating PBLs, APCs and DCs are well known, and the epitopemay be delivered to the APCs in vitro in any form, including by directlydepositing the epitope on the cells, or by liposome or exosome delivery,as described herein, alone or in the presence of additional factors,such as heat shock proteins or appropriate cytokines.

Recent reports suggest that cross-linking of EphA2 on the cell surfaceof tumor cells by a ligand agonist provokes EphA2 phosphorylation,internalization and degradation (Walker-Daniels et al., Mol. Cancer.Res. 1: 79-87, 2002). This triggered degradation of EphA2 protein isbelieved to result in the acute generation of EphA2 epitope presented byMHC class I and/or class II proteins, making the tumor more easilyrecognized by EphA2-specific T cells, and potentially resulting inimproved clinical eradication of cancer cells in vivo. This supports theconcerted use of EphA2-based vaccines to expand and activate effectoranti-EphA2 T cells in cancer patients with EphA2-ligand agonists toincrease the likelihood for productive recognition of tumor cells byvaccine-induced lymphocytes in combinational immunotherapy approaches.EphA2 ligand agonists could take the form of, but would not berestricted to, anti-EphA2 antibodies, EphrinA1-Ig constructs orsynthetic peptides that induce degradation of EphA2 protein in treatedtumor cells.

In one embodiment, a patient is administered an EphA2 ligand or anagonist thereof. The EphA2 ligand or agonist thereof can be a bindingreagent, such as an antibody (for example a monoclonal antibody, or aderivative or an analog of an antibody, including without limitation: Fvfragments; single chain Fv (scFv) fragments; Fab′ fragments; F(ab′)2fragments; camelized antibodies and antibody fragments; multivalentversions of the foregoing; monospecific or bispecific antibodies, suchas disulfide stabilized Fv fragments, scFv tandems ((scFv)₂ fragments),antibody multimers, which typically are covalently linked or otherwisestabilized (for example and without limitation, leucine zipper or helixstabilized); scFv fragments; recombinant antibodies or antibodyfragments), or in vitro-generated EphA2-specific compounds, such asaptamers and compounds generated by phage display selection andpropagation methods. The EphA2 ligand or agonist thereof can be ephrinA1or an agonist thereof.

The EphA2 ligand or agonist thereof can be delivered to a patient by anyeffective route, in any effective amount and by any effective interval,as described above in reference to the EphA2 T-cell receptor epitope.Delivery of the EphA2 ligand or agonist thereof is in combination withthe delivery of the EphA2 T-cell receptor epitope-containing compounds,and a therapeutic regimen typically, but not necessarily alternatesdelivery of the EphA2 T-cell receptor epitopes and the EphA2 ligand oragonist thereof. In one embodiment, the EphA2 T-cell receptor epitopesare delivered to a patient either directly by the direct or ex vivomethods described above, and one week later, the EphA2 ligand or agonistthereof is administered. This is repeated any desired and effectivenumber of times, with one treatment per week, alternating between theEphA2 T-cell receptor epitopes and the EphA2 ligand or agonist thereof.

Any peptide described herein can be manufactured by any of the myriad ofknown recombinant methods for producing a peptide, or can be synthesizedby common protein synthesis methods, such as by solid phase chemistriesas are broadly known. In a recombinant method, a gene is prepared thatcontains an appropriate promoter, terminator and a coding region thatencodes the desired peptide. The coding region also can encode signalsequences enabling secretion of the peptide by cells and/or suitabletags, preferably cleavable, that permit affinity purification of thepeptide.

A nucleic acid containing a gene for expressing an EphA2 T-Cell receptorepitope in a human cell also may be delivered directly to a patient, orex vivo to a patient's cells (for delivery back into the patient) suchthat the cells of the patient express the epitope, thereby eliciting animmune response. Delivery of a gene, rather than just the epitope canresult in a more robust immune response resulting from the extendedexpression of the gene in the patient's cells. The nucleic acid can bedelivered by viral-mediated (such as by a recombinant adenovirus,adeno-associated virus or vaccinia virus) or non-viral-mediated deliverymethods (such as by liposome or by direct injection of naked nucleicacid, for instance into muscle).

EXAMPLES Example 1 Reactivity of PBLs Against EphA2 T-Cell PeptideEpitopes

Peripheral Blood and Tumor Specimens. Peripheral blood samples wereobtained by venipuncture from 40 patients diagnosed with RCC and 14normal individuals and were collected into heparinized tubes. Peripheralblood lymphocytes (PBLs) were isolated by centrifugation on aFicoll-Hypaque gradient (LSM, Organon-Teknika, Durham, N.C.). RCC tumorlesions and matched normal kidney tissue were surgically-resected andparaffin-embedded. Informed consent under an IRB-approved protocol wasobtained from all patients prior to sample acquisition. Patient andnormal donor information is provided in Table 1. All individualsincluded were HLA-A2 positive or/and HLA-DR4 positive, as determined byfluorescence-activated cell sorter analysis using the HLA-A2-specificantibodies (BB7.2 and MA2.1) and HLA-DR4-specific antibody (anti-HLA-DR4monoclonal antibody clone 359-13F10, IgG, kindly provided by Dr. JaniceBlum, Indiana University School of Medicine, Indianapolis, Ind.). Amongthe RCC patients and normal individuals, 9 patients and 6 normalindividuals expressed both the HLA-A2 and HLA-DR4 majorhistocompatibility antigens.

TABLE 1 HLA-A2 and/or DR4 positive RCC patients evaluated Disease statusat RCC time of evaluation HLA Typing: Patient Age Sex Stage Treatment(Months) A2(+/−) DR4(+/−) SLR30-pre 63 F I none Local Dis. + − SLR31 66M I none Local Dis. + − SLR32 62 F I none Local Dis. + − SLR33 54 F Inone Local Dis. + − SLR34 71 M I none Local Dis. + + SLR35 75 F I noneLocal Dis. + + SLR36-pre 60 M I none Local Dis. + + SLR37 52 M I noneLocal Dis. + − SLR38-pre 69 M I none Local Dis. + − SLR39 65 M I S NED(3) + − SLR30-post 63 F I S NED (1.5) + − SLR40 53 M I S NED (3) + −SLR36-post 60 M I S NED (2) + + SLR41 64 F I S NED (2) + − SLR38-post 69M I S NED (2) + − SLR42 58 F I S Local Dis. (3) + − SLR43 53 F I S LocalDis. (1.5) + − SLR44-pre 69 M IV none Mets + − SLR45 65 M IV none Mets +− SLR46 45 F IV none Mets + − SLR47 53 F IV S NED (1.5) + − SLR48 54 MIV S Mets (61) + − SLR49 52 F IV S, R, IFN-α, IL-2 Mets (41) + −SLR44-post 69 M IV S Mets (2) + − SLR50 54 M IV S, R, C Mets (21) + −SLR51 41 M IV S, R, IL-2 Mets + + SLR52 58 M IV S, R, IFN-α Mets + +SLR53 52 M IV S Mets + − SLR54 49 F IV IL-2, C Mets + + SLR55 79 M IVIFN-α, C Mets + + SLR56 56 M IV R, IL-2, IFN-α, C Mets + − SLR57 68 F IVnone Mets + − SLR58 55 F IV none Mets + + SLR59 52 F I none Local Dis.− + SLR60-pre 58 M I none Local Dis. − + SLR61 60 M I none Local Dis.− + SLR62 64 M I S NED (3) − + SLR63 53 F I S NED (1.5) − + SLR60-post58 M I S NED (2) − + SLR64 65 M I S NED (10) − + SLR65 53 M II S LocalDis. − + SLR66 45 M IV none Mets − + SLR67 57 M IV C, R Mets − + SLR6869 M IV S, R, C Mets − + SLR69 49 M IV S, C, R, IL-2, IFN-α Mets − +

In Table 1, individual CCF designations reflect specimen number based ondate harvested. In 5 cases, both pre- and (6 weeks) post-therapy bloodspecimens were available for analysis, as indicated. Where indicated,the time of peripheral blood isolation (in months) post-therapy isprovided. Abbreviations used: C, Chemotherapy; IFN-γ, recombinantInterferon-alpha therapy; IL-2, recombinant Interleukin-2 therapy; Mets,Metastatic Disease; NED, No evidence of disease; R, Radiotherapy; S,Surgery. HLA-A2 and -DR4 status was determined using allele-specificmonoclonal antibodies and flow cytometry gating on peripheral bloodmonocytes, as described in Materials and Methods.

Cell Lines and Media. The T2.DR4 (HLA-A2^(+/−)DRβ1*0401⁺; Pratt, R. L.et al. Oncogene 21:7690-7699 (2002)) cell line (kindly provided from Dr.Janice Blum, Indiana University School of Medicine, Indianapolis, Ind.)was used as the peptide-presenting cell in ELISPOT assay. The followingSLR20-SLR26 clear cell RCC lines were evaluated in Western Blottinganalyses. The normal human proximal tubular epithelial kidney cell lineHK-2 (American Type Tissue Collection, ATCC, Rockville, Md.) was alsoevaluated in these analyses. Hypothetically, HK-2 represents a normalcontrol cell line, although it has been transformed by transfection withthe HPV-16 E6/E7 genes (Ryan M J, et al. Kidney Int. 45:48-57 (1994)).The EphA2+ PC-3 prostate carcinoma cell line was included as a positivecontrol for Western blotting (Walker-Daniels J, et al. Prostate41:275-280 (1999)). All cell lines were maintained in RPMI-1640 culturemedium supplemented with 10% heat-inactivated fetal bovine serum, 100U/ml penicillin, 100 μg/ml streptomycin and 10 mM L-glutamine (allreagents from GIBCO/Life Technologies, Grand Island, N.Y.) in ahumidified atmosphere of 5% CO₂ at 37° C.

Peptides selection and synthesis. The protein sequence of EphA2 proteinwas obtained from GENBANK (accession number AAH37166; FIG. 1) andanalyzed for HLA-A0201 and HLA-DR.beta.1*0401 binding peptides usingneural network algorithms (Honeyman M C, Brusic V, Stone N L, Harrison LC., “Neural network-based prediction of candidate T-cell epitope,” NatBiotechnol. (1998) 16:966-969 and Southwood S, Sidney J, Kondo A, delguercio M-F, Appella E, Hoffman S, Kubo R T, Chesnut R W, Grey H M,Sette A., “Several common HLA-DR types share largely overlapping peptidebinding repertoires,” J. Immunol. (1998) 160:3363-3383). The top tencandidate HLA-A2 binding peptides were then analyzed for their abilityto be generated by proteasomal cleavage using the PAProC predictionalgorithm (C. Kuttler, A. K. Nussbaum, T. P. Dick, H.-G. Rammensee, H.Schild, K. P. Hadeler, “An algorithm for the prediction of proteasomalcleavages,” J. Mol. Biol. (2000) 298:417-429; A. K. Nussbaum, C.Kuttler, K. P. Hadeler, H.-G. Rammensee, H. Schild, PAProC: A PredictionAlgorithm for Proteasomal Cleavages available on the WWW, Immunogenetics53 (2001), 87-94; and A. K. Nussbaum, “From the test tube to the WorldWide Web—The cleavage specificity of the proteasome,” dissertation,University of Tuebingen, Germany, 2001(www-dot-uni-tuebingen-dot-de-slash-uni-slash-kxi-slash, with only thosepeptides capable of being processed by the proteasome selected forsynthesis. All peptides were synthesized by Fmoc chemistry. Peptideswere >90% pure based on HPLC profile and MS/MS mass spectrometricanalysis. In total, five HLA-0201 and three HLA-DR0401 predicted bindingpeptides that received high binding scores in this study (Table 2), wereevaluated.

TABLE 2 Selection of EphA2 Peptides for Analysis Selected HLA-A2Presented EphA2 Peptides: Selected Start AA Sequence ProteasomeSynthesized Amino Acid of Nonamer Binding Score* Generated? For Analysis883 TLADFDPRV¹ 1084   YES YES 546 VLLLVLAGV² 1006   YES YES 550VLAGVGFFI³ 556  NO NO  58 IMNDMPIYM⁴ 138  NO NO 961 SLLGLKDQV⁵ 127  YESYES 253 WLVPIGQCL⁶ 98 NO NO  12 LLWGCALAA⁷ 71 NO NO 391 GLTRTSVTV⁸ 70YES YES 120 NLYYAESDL⁹ 68 NO NO 162 KLNVEERSV¹⁰ 49 YES YES *The higherthe binding score, the greater the stability of the predictedpeptide-MHC complex. Binding scores and qualitative determination ofproteasomal processing were predicted using on-line algorithms asdescribed in Materials and Methods. ¹SEQ ID NO: 2, residues 883-891.²SEQ ID NO: 2, residues 546-554. ³SEQ ID NO: 2, residues 550-558. ⁴SEQID NO: 2, residues 58-66. ⁵SEQ ID NO: 2, residues 961-969. ⁶SEQ ID NO:2, residues 253-261. ⁷SEQ ID NO: 2, residues 12-20. ⁸SEQ ID NO: 2,residues 391-399. ⁹SEQ ID NO: 2, residues 120-128. ¹⁰SEQ ID NO: 2,residues 162-170. Selected HLA-DR4 Presented EphA2 Peptides: SequenceStart AA Sequence Core AA # of Nonamer Binding Score Synthesized ForAnalysis 666  IMGQFSHHN¹ 577 ₆₆₃EAGIMGQFSHHNIIR² 67 YSVCNVMSG³ 95₆₃PIYMYSVCNVMSG⁴ 55 MQNIMNDMP⁵ 39 ₅₃DLMQNIMNDMPIYMYS⁶ ¹SEQ ID NO: 2,residues 666-674. ²SEQ ID NO: 2, residues 663-677. ³SEQ ID NO: 2,residues 67-75. ⁴SEQ ID NO: 2, residues 63-75. ⁵SEQ ID NO: 2, residues55-63. ⁶SEQ ID NO: 2, residues 53-68.

Antigen Stimulation of PBLs. PBLs were resuspended at 10⁷/ml in AIM-Vmedium (GIBCO/Life Technologies) and were incubated for 60 min at 37° C.in a humidified 5% CO₂ incubator. Nonadherent (T cell-enriched) cellswere gently washed out with PBS and subsequently frozen. The plasticadherent cells were cultured in AIM-V medium supplemented with 1000units/ml rhGM-CSF (Immunex Corporation, Seattle, Wash.) and 1000units/ml rhIL-4 (Schering-Plough, Kenilworth, N.J.). Seven days later,dendritic cells (DCs) were harvested and used to stimulate autologousCD8⁺ or CD4⁺ T cells. Non-adherent autologous cells were used as“enriched” sources of T cell responders. CD8⁺ T cells (inHLA-A2-positive patients and healthy donors) or CD4⁺ T cells (inHLA-DR4-positive patients and healthy donors) were positively isolatedto >98% purity using specific magnetic beads (MACS; Miltenyi Biotec,Auburn, Calif.). Two hundred thousand DCs were cocultured with 2×10⁶CD8⁺ or CD4⁺ T cells with 10 μg/ml peptide for 1 week. On day 7 of invitro stimulation, the responder CD8⁺ T cells or CD4⁺ T cells wereharvested and analyzed in ELISPOT assays.

IFN-γ and IL-5 ELISPOT assays for Peptide-Reactive CD8⁺ T cells and CD4+T cell Responses. To evaluate the frequencies of peripheral blood Tcells recognizing peptide epitopes, ELISPOT assays for IFN-γ and IL-5were performed, as previously described (Tatsumi T, et al. J. Exp. Med.196; 619-628, 2002). CD8⁺ T cell responses were evaluated by IFN-γELISPOT assays only, while CD4⁺ T cell responses were evaluated by bothIFN-γ (Th1) and IL-5 (Th2) ELISPOT assays. For ELISPOT assays, 96-wellmultiscreen hemagglutinin antigen plates (Millipore, Bedford, Mass.)were coated with 10 μg/ml of antihuman IFN-γ mAb (1-D1K; Mabtech,Stockholm, Sweden) or 5 μg/ml of antihuman IL-5 (Pharmingen-BD, SanDiego, Calif.) in PBS (GIBCO/Life Technologies) overnight at 4° C.Unbound antibody was removed by four successive washing with PBS. Afterblocking the plates with RPMI-1640/10% human serum (1 hr at 37° C.), 10⁵CD8+ T cells or CD4+ T cells and T2.DR4 cells (2×10⁴ cells) pulsed with10 μg/ml synthetic peptides were seeded in triplicates in multi-screenhemagglutinin antigen plates. Control wells contained CD8+ or CD4+ Tcells with T2.DR4 cells pulsed with HIV-nef₁₉₀₋₁₉₈ peptide (AFHHVAREL,SEQ ID NO: 3) or Malaria-CS₃₂₆₋₃₄₅ peptide (EYLNKIQNSLSTEWSPCSVT; SEQ IDNO: 4), respectively, or T2.DR4 cells alone. Culture medium was AIM-V(GIBCO/Life Technologies) at a final volume of 200 μl/well. The plateswere incubated at 37° C. in 5% CO₂ for 24 hr for IFN-γ assessments, and40 hr for IL-5 assessments. After incubation, the supernatants of theculture wells were harvested for ELISA analyses, and cells were removedby washing with PBS/0.05% Tween 20 (PBS/T). Captured cytokines weredetected at sites of their secretion by incubation for 2 hr withbiotinylated mAb anti-human IFN-γ (7-B6-1; Mabtec) at 2 μg/ml inPBS/0.5% BSA or biotinylated mAb anti-human IL-5 (Pharmingen) at 2 μg/mlin PBS/0.5% BSA. Plates were washed six times with PBS/T, andavidin-peroxidase complex (diluted 1:100; Vectastain Elite Kit; VectorLaboratories, Burlingame, Calif.) was added for 1 hr. Unbound complexwas removed by three successive washes with PBS/T, then with threerinses with PBS alone. AEC substrate (Sigma, St. Louis, Mo.) was addedand incubated for 5 min for the IFN-γ ELISPOT assay and the TMBsubstrate for peroxidase (Vector Laboratories) was added and incubatedfor 10 min for the IL-5 ELISPOT assay. Spots were imaged using the ZeissAutolmager (and statistical comparison determined using a Studenttwo-tailed T-test analysis). The data are represented as mean IFN-γ orIL-5 spots per 100,000 CD4+ T cells analyzed.

ELISAs. The supernatants harvested from CD4+ T cell ELISPOT plates wereanalyzed in TGF-β and IL-10 ELISAs. Supernatants were isolated fromELISPOT plates at the endpoint of the culture period and frozen at −20°C. until analysis in specific cytokine ELISAs. Cytokine capture anddetection antibodies and recombinant cytokine were purchased fromBD-Pharmingen (San Diego, Calif.) and used in ELISA assays per themanufacturer's instructions. The limit of detection for the TGF-β andIL-10 assays was 60 pg/ml and 7 pg/ml, respectively.

Western blot analysis. Tumor cells (5-10×10⁶) were analyzed for EphA2expression via Western blots using the anti-human EphA2 polyclonalantibody (clone: H-77) (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif.). Cell pellets were lysed using 200 μl of 1% NP-40 in PBScontaining protease inhibitors (Complete, Boehringer Mannheim,Indianapolis, Ind.) for 1 hour on ice. After centrifugation at 13,500×gfor 30 minutes, the supernatant was mixed 1:1 with SDS-PAGE runningbuffer and proteins separated on 10% PAGE gels, prior toelectro-blotting onto nitrocellulose membranes (Millipore, Bedford,Mass.). Blots were imaged on Kodak X-Omat Blue XB-1 film (NEN LifeScience Products, Boston, Mass.) using horseradish peroxidase(HRP)-conjugated goat anti-rabbit Ig (Biorad, Hercules, Calif.) and theECL chemiluminescence detection kit (NEN Life Science Products).

Immunohistochemistry for EphA2 in RCC tissue. RCC tumor specimens wereobtained surgically under an IRB-approved protocol andparaffin-embedded. Five μm sections were de-paraffinized and rehydratedin ddH₂O and then PBS. Anti-EphA2 mAb (Ab 208; mIgG1) or isotype-matchedcontrol mAb was incubated on sections for 1 h at RT. After PBS washing,sections were incubated with biotinylated goat anti-rabbit IgG (VectorLaboratories) for 20 min at room temperature, and after washing, werethen incubated with avidin-biotin-complex peroxidase (Vectastain ABCkits, Vector Laboratories). After a subsequent wash, reaction productswere developed by Nova Red substrate kit (Vector Laboratories), andnuclei were counterstained with hematoxylin. The expression of EphA2 wasevaluated independently by two investigators with a microscope under 40×magnification.

Statistical Analysis. Statistical significance of differences betweenthe two groups was determined by applying Student's t test or two samplet test with Welch correction after each group had been tested for equalvariance. Statistical significance was defined as a p value of less than0.05.

Results

Expression of EphA2 in tumor cell lines and in RCC tissues. EphA2 wasoverexpressed in malignant renal epithelial cells. Western blot analyseswere used to evaluate EphA2 protein levels in RCC cell lines (FIG. 18).Metastatic RCC lines tended to express EphA2 more strongly than primaryRCC lines, approaching the strong staining previously noted for theprostate carcinoma PC-3 (Walker-Daniels J, et al. Prostate 41; 275-280,1999). While used as a model for normal proximal kidney endothelialcells, the HK-2 cell line is HPV-16 E6/E7-transformed and expresseslevels of EphA2 consistent with that observed for primary RCC lines.Normal PBLs failed to express detectable levels of EphA2 protein.Consistent with these findings, immunohistochemical analyses performedon paraffin-embedded RCC specimens (FIG. 19) verified strong expressionof EphA2.

In FIG. 18, anti-EphA2 and control anti-β-actin antibodies were used inperforming Western Blot analyses of lysates generated from the indicatedRCC cell lines, the normal kidney tubular epithelial cell line HK2 andnormal peripheral blood lymphocytes (PBLs) (negative control). Primaryand metastatic clear cell RCC lines were assessed, as indicated. The PC3prostate cell line and normal donor PBLs served as positive and negativecontrols. In FIGS. 19A-D, primary (FIGS. 19A and 19B) and metastatic(FIGS. 19C and 19D) RCC paraffin tissue sections were stained usinganti-EphA2 antibody (Ab 208; FIGS. 19A and 19C) or isotype controlantibody (FIGS. 19B and 19D) in immunohistochemical analyses (40×magnification).

Identification of EphA2 epitopes recognized by T cells. To identifypotential T cell epitopes, the EphA2 protein sequence was subjected toalgorithms designed to identify putative HLA-A2 binding motifs and sitesof proteasomal cleavage. Similarly, a neural network algorithm was usedto identify EphA2 peptide sequences that would be predicted to bindHLA-DR4 and have the potential to represent CD4⁺ T cell epitopes(Honeyman M et al., 1998). In aggregate, 8 peptides were synthesized forsubsequent analyses: 5 peptides were predicted to serve as CTL epitopesand 3 peptides were predicted to serve as Th epitopes (Table 2).

Peripheral blood T cells were isolated from normal HLA-A2⁺ and/or -DR4⁺donors and stimulated with autologous DCs that had be previously loadedwith relevant synthetic peptides. Responder T cells were subsequentlyevaluated for specific reactivity against peptide-pulsed T2.DR4(HLA-A2⁺/DR4⁺) antigen-presenting cells and renal cell carcinoma celllines that expressed both the EphA2 antigen and HLA-A2 and/or HLA-DR4.The IFN-γ ELISPOT assay was used to evaluate 8 HLA-A2⁺ donor CD8⁺ T cellresponses to the 5 putative CTL epitopes and 7 HLA-DR4+ donor CD4⁺ Tcell reactivities against the 3 potential Th epitopes.

Each peptide was recognized by at least one normal donor (Table 3), andonly one (HLA-DR4⁺) donor failed to respond to any of the EphA2 (Th)epitopes. Among the HLA-A2 donors, the EphA2₅₄₆₋₅₅₄ and EphA2₈₈₃₋₈₉₁peptides were most commonly reacted against (each in 6/8 donorsevaluated), with the responses to EphA2₈₈₃₋₈₉₁ typically being of ahigher frequency. Among the HLA-DR4⁺ donors evaluated, 6/7 donorsresponded against at least one predicted EphA2-derived Th epitope, withresponses against the EphA₆₃₋₇₅ and EphA2₆₆₃₋₆₇₇ most prevalent. Whencloned T cells were derived from these bulk populations of responder Tcells, they were capable of recognizing EphA2⁺ RCC lines in theappropriate HLA class I- or class II-(HLA-A2 or -DR4) restricted manner(data not shown).

TABLE 3 Normal donor T cell responses to putative EphA2-derived peptideepitopes HLA-A2-Presented EphA2 Peptides: CD8+ T Cell Response toPeptide on T2.DR4^(a): Normal Donor # 162  391  546  883 961  A2-1  9  0^(b) 31  0  2 A2-2 40 81 14  85 21 A2-3  3 14 10  0 21 A2-4  2  0 11 58  0 A2-5 11  0 14 172  4 A2-6  0 91 76 145 13 A2-7 132   0  0  37  0A2-8 15  0  0 165  0 Total Responses: 5/8 3/8 6/8 6/8 3/8HLA-DR4-Presented EphA2 Peptides: CD4+ T Cell Response to Peptide onT2.DR4^(a): Normal Donor # 53 63 663 DR4-1 43 11 21 DR4-2 38 36 57 DR4-3 4  7 14 DR4-4  0  0  0 DR4-5  0 156  41 DR4-6  0 121  67 DR4-7 54 48 72Total Responses: 3/7 6/7 6/7 ^(a)T cell responses over T2.DR4 pulsedwith control peptides/100,000 T cells. ^(b)A value of “0” reflects afrequency <1/100,000 T cells. T cell reactivity against T2.DR4 cellspulsed with the HLA-A2-presented HIV-nef₁₉₀₋₁₉₈ epitope served as theCD8⁺ T cell negative control, while HLA-DR4-presented Malarialcircumsporozooite (CS)₃₂₆₋₃₄₅ epitopeserved as the CD4⁺ T cell negativecontrol. These control values were subtracted from experimentaldeterminations in order to determine EphA2-specific T cell # responderspot numbers. Values significantly (p < 0.05) elevated over T2.DR4⁺control peptide values are underlined.

Analysis of peptide-specific IFN-γ release by peripheral blood CD8⁺ Tcells in ELISPOT assays. Peripheral blood CD8⁺ T cells responses wasassessed against these sequences in 29 HLA-A2⁺ RCC patients (Table 1)and 10 HLA-A2⁺ normal donors. CD8⁺ T cells were enriched to 98% purityfor all experiments. Responses were evaluated using IFN-γ ELISPOT assaysafter 7 day “primary” in vitro stimulations.

In FIG. 20, peripheral blood CD8⁺ T cells were isolated from HLA-A2⁺normal donors or patients with RCC and stimulated with immature,autologous dendritic cells pre-pulsed with the individual EphA2-derivedepitopes, as outlined in Materials and Methods. After one week,responder T cells were analyzed in IFN-γ ELISPOT assays for reactivityagainst T2.DR4 (HLA-A2⁺) cells pulsed with the indicated EphA2 epitope.Data are reported as IFN-γ spots/100,000 CD8⁺ T cells and represent themean of triplicate determinations. T cell reactivity against T2.DR4cells pulsed with the HLA-A2-presented HIV-nef₁₉₀₋₁₉₈ epitope served asthe negative control in all cases, and this value was subtracted fromall experimental determinations in order to determine EphA2-specificspot numbers. Each symbol within a panel represents an individualdonor's response.

As shown in FIG. 20, the frequencies of CD8⁺ T cell responses againstEphA2 peptides in HLA-A2⁺ patients prior to surgery (Pre-Op) or patientswith residual disease after surgery (Post-RD) were as low as thoseobserved in normal HLA-A2⁺ donors. In contrast, elevated ELISPOTreactivity to EphA2 epitopes was observed in RCC patients who werecategorized as disease-free (no-evidence of disease: NED) after surgery(Post-NED). Interestingly, CD8⁺ T cells from RCC patients exhibitinglong-term survival (Post-LTS; >2 year survival post-surgery) despitehaving some degree of active disease, also showed elevated ELISPOTreactivity to EphA2 CTL epitopes. There were no significant differencesin anti-EphA2 CD8⁺ T cell responses when comparing patients with Stage Ivs. Stage IV, if the patient had active disease (FIG. 21, showing datareported in FIG. 20 re-plotted as a function of disease-stage). Onlypatients that were analyzed at a time when they were disease-free (i.e.no evidence of disease, NED) or if they were long-term survivors,exhibited CD8⁺ T cells with elevated reactivity to EphA2 epitopes (FIG.21).

The change of CD8⁺ T cell reactivity against EphA2 peptides pre- andpost-therapy in 4 HLA-A2⁺ patients was evaluated. In FIG. 22, Peripheralblood CD8⁺ T cells were isolated pre- and (6 week) post-surgery frompatients with RCC, and evaluated for reactivity to EphA2 epitopes inIFN-γ ELISPOT assays, as outlined in the FIG. 20 description, above. Thethree Stage I RCC patients (●, ◯, ▾) were rendered free of disease as aresult of surgical intervention, while the single Stage IV RCC patient(∇) had residual disease after surgery. Each symbol within a panelrepresents an individual patient's response. Three of these individualswere Stage I patients who had local disease prior to surgicalintervention, while the remaining patient had Stage IV disease. Notably,CD8⁺ T cell reactivity against EphA2 peptides was very low prior tosurgery in all four RCC patients. After being rendered free of disease,CD8⁺ T cell reactivity against EphA2-derived CTL epitopes wassignificantly increased in each of the three Stage I patients. In markedcontrast, the single evaluable Stage IV RCC patient, who had residualtumor burden after surgery, remained poorly responsive to EphA2 peptides(FIG. 22).

Peptide-specific IFN-γ and IL-5 release by CD4⁺ T cells in ELISPOTassay. IFN-γ (Th1-type) and IL-5 (Th2-type) ELISPOT assays were used todiscern altered frequency and functional bias of patient-derived Thcells against EphA2 peptides. Peripheral blood T cells were stimulatedfor one week with peptide-pulsed immature autologous DC (which do notappear to skew the Th1/Th2 balance, ref. 47) prior to CD4⁺ T cellisolation and ELISPOT analyses. The frequencies of CD4⁺ T cellresponders against EphA2 peptides were evaluated in 19 HLA-DR4⁺ RCCpatients (Table 1).

The functional nature of T cell reactivity towards EphA2 related todisease progression. Patients with Stage I disease patients displayedstrongly Th1-polarized reactivity against EphA2 peptides whereaspatients with more advanced stages of the disease polarized towardsstrong Th2 reactivity. In FIG. 23, peripheral blood was obtained from 19HLA-DR4⁺ patients (Table 1) and CD4⁺ T cells isolated by positiveMACS™.-bead selection as described in Materials and Methods, below.After a one-week in vitro stimulation with EphA2 Th peptide-pulsed,autologous DCs, responder CD4⁺ T cells were evaluated against T2.DR4cells pulsed with the indicated EphA2 epitopes in IFN-γ and IL-5 ELISPOTassays. Data are reported as IFN-γ spots/100,000 CD4⁺ T cells andrepresent the mean of triplicate determinations. T cell reactivityagainst T2.DR4 cells pulsed with the HLA-DR4-presented Malarialcircumsporozooite (CS)₃₂₆₋₃₄₅ epitope served as the negative control inall cases, and this value was subtracted from all experimentaldeterminations in order to determine EphA2-specific spot numbers. Eachsymbol within a panel represents an individual patient's response. Notevery patient reacted against each peptide, but their responses wereconsistently polarized in accordance with the patient's disease stage.

One set of matched blood samples from an HLA-DR4⁺ patient pre- andpost-therapy fortunately was available. This individual had beenrendered free of disease after surgery. While the CD4⁺ T cells from thisdonor were Th1-biased before and after surgery, the frequency of IFN-γspots associated with T cell responses against the EphA2₅₃₋₆₈ andEphA2₆₃₋₇₅ (but not the EphA2₆₆₃₋₆₇₇) epitopes increased post-treatment.In FIG. 24, pre- and post-surgery peripheral blood was available for asingle RCC patient with Stage I disease. CD4⁺ T cells were isolated andanalyzed for reactivity to EphA2 Th epitopes, as outlined in the FIG. 23description, above. A statistically-significant increase in Th1-type(IFN-γ) and decrease in Th2-type (IL-5) CD4⁺ T cell responsepost-surgery was noted for the EphA2₅₃₋₆₈ epitope. Therapy-inducedchanges in CD4⁺ T cell response to the EphA2₆₃₋₇₅ epitope were similar,with the IFN-γ results approaching a p value of 0.05 and the significantreductions in IL-5 responses noted (p<0.001). T cell responses to theEphA2₆₆₃₋₆₇₇ epitope pre-/post-sugery were not significantly different.The ratio of Th1/Th2-type responses pre- and post-therapy is alsoindicated for peptides EphA2₅₃₋₆₈ and EphA2₆₃₋₇₅. p values forsignificant differences are indicated.

This donor was also HLA-A2 and it was observed that increased Th1-typeCD4⁺ T cell-mediated immunity to EphA2 occurred in concert withincreased frequencies of circulating IFN-γ-secreting anti-EphA2 CD8⁺ Tcells in this patient (FIG. 22; filled circles).

TGF-β and IL-10 production from RCC patient CD4⁺ T cells against EphA2peptides. To evaluate whether Th3/Tr1 CD4⁺ T cells were present in theperipheral blood of RCC patients, TGF-β and IL-10 production followingin vitro peptide-stimulation was measured. In FIG. 25, supernatants wereharvested from the culture wells of IFN-γ ELISPOT assays and analyzedfor levels of TGF-β1 using a commercial ELISA procedure. Of 19 HLA-DR4patients evaluated, only the supernatants of 3 (of 8 evaluated) patientswith Stage IV RCC contained detectable quantities of TGF-β1. Thecorresponding IFN-γ and IL-5 ELISPOT data for these patients' CD4+ Tcells is also provided. Each symbol within a panel represents anindividual patient's response. TGF-β production by responder CD4⁺ Tcells was only observed in a subset (i.e. 3 of 8) of Stage IV patientsand notably, these same patients displayed coordinately weak Th1- orTh2-type (IFN-γ and IL-5 ELISPOT) CD4⁺ T cell reactivity against EphA2peptides. IL-10 production (above the detection limit of the ELISA) wasnot observed for any specimen tested.

The molecular definition of tumor-associated antigens has facilitatedthe development of immunotherapies designed to prime and boosttumor-specific T cell responses in cancer patients. In concert withthese advances, cytokine release assays provide a powerful means tomonitor the specificity and magnitude of evolving anti-tumor CD8⁺ andCD4⁺ T cell responses in the peripheral blood of patients before, duringand after treatment (Keilholz U, et al. J Immunother 25; 97-138, 2002).In the present example, how, and to what extent, T cells in patientswith RCC recognize novel EphA2-derived epitopes was evaluated usingcytokine-specific ELISPOT assays and ELISAs.

The major finding of this example is a demonstration that renal cellcarcinoma patients exhibit detectable CD4⁺ and CD8⁺ T cell reactivitytowards the receptor tyrosine kinase EphA2 that is aberrantly expressedat a high frequency in RCC tumors. EphA2-specific CD8⁺ T cell activityis inversely proportional to the presence of active disease in thesepatients and is increased within 6 weeks following therapeuticintervention that results in disease-free status. Interestingly, twoHLA-A2⁺ patients with Stage IV disease were identified who werelong-term survivors (>40 months) post-surgery. Both of these individualsdisplayed elevated peripheral blood frequencies of IFN-γ-secreting CD8⁺T cells reactive against EphA2-derived epitopes (FIG. 20). Continuedmaintenance of high anti-EphA2 CD8⁺ T cell activity in these patientsmay relate to their continued survival with active disease.

Somewhat in contrast with the CD8⁺ T cell results, it is also shownherein that a fine balance of patient Th1-type versus Th2-type CD4⁺ Tcell responses to EphA2 peptides distinguishes between disease-grades.In particular, the most advanced forms of RCC tend to polarize towardsTh2- or Tr-type anti-EphA2 responses. This polarization in functionalCD4⁺ T cell responsiveness, combined with the potential suppressiveactivity mediated by T regulatory cells in patients with Stage IVdisease, may play facilitating roles in disease progression.

These findings are unique in part because they indicate that that EphA2may provide a much-needed target antigen for the design ofimmunotherapies for renal cell carcinoma. First, EphA2 is over-expressedin a large number of RCC specimens, including 22 of 24 (92%) RCC celllines and 29 of 30 (97%) RCC biopsy samples, respectively (FIG. 18 anddata not shown). These findings are consistent with evidence emergingfrom studies of other tumor types, which indicates that high levels ofEphA2 apply to many cancers, including breast, colon, head and neck(Tatsumi et al., unpublished data), prostate and lung carcinoma, as wellas, melanoma. If the present studies can be extended to these otherclinical indications, EphA2-specific T cell activity could provide anopportunity for therapeutic intervention for these tumor types as well.

Interestingly, CD8⁺ T cell reactivity against EphA2 peptides (asdetermined in IFN-γ ELISPOT assays) differed greatly between RCCpatients with active disease and those patients rendered free ofdisease. Yet, anti-EphA2 CD8⁺ T cell reactivity did not distinguish RCCdisease stage. One potential explanation for this finding is that RCCtumors may suppress the generation, functionality and durability of CD8⁺T cell responses against EphA2 in situ. This hypothesis is consistentwith general tumor-associated immune suppression of peripheral CTL andNK cell activity, as has been previously reported (Kiessling R, et al.Cancer Immunol Immunother 48; 353-362, 1999). Notably, CD8⁺ T cellreactivity against EphA2-derived CTL epitopes significantly increased inthe peripheral blood of three HLA-A2⁺ patients with Stage I RCC aftersurgery that rendered these individuals free of disease. In contrast, ina Stage IV patient, surgical intervention without “cure” did not changethe low frequency of CD8⁺ T cell reactivity towards EphA2 peptides.These results are consistent with the requirement for RCC tumorclearance in situ (That is, termination of chronic (tumor) antigenicstimulation) to allow for elevation in functional Tc1-like anti-tumorCD8⁺ T cell responses (Liu H, et al. J Immunol 168:3477-3483, 2002 andMoser J M, et al. Viral Immunol 14:199-216, 2001). An alternativeexplanation is that expansion or maintenance of EphA2-specific CD8⁺ Tcell activity may require the concerted support of specific Th1-typeresponses or a shift of existing patient Th2-type or T suppressor-typeto Th1-type immunity, particularly in the advanced cancer setting(Tatsumi et al., J. Exp. Med. 2002).

Th1-type biased CD4⁺ T cell response could only be observed in a subsetof Stage I RCC patients, and Th2- or Tr-type biased CD4⁺ T cellresponses were almost always observed in Stage IV RCC patients. It isimportant to stress that polarization away from Th1-type immunity inpatients with advanced stage disease was tumor-specific, sinceindividuals with Stage IV disease responded to influenza- andEBV-derived T helper epitopes in a “normal” Th1-biased manner (Tatsumiet al., J. Exp. Med. 2002 and data not shown).

While longitudinal data was available for only one HLA-DR4⁺ patient withStage I disease (FIG. 24), Th1-type immunity against at least some EphA2epitopes was strengthened and EphA2-specific, Th2-type responseslessened after surgical resection of the patient's tumor. These resultsare consistent with previous reports that in most cancers, the immuneresponse is believed to be suppressed (or deviated) in advanced stagecancer patients. These results also suggest that the nature of CD4⁺ Tcell responses against “late-stage” EphA2 peptides correlates with RCCdisease stage. This finding contrasts with these previous observationsfor CD4⁺ T cell responses against the “early-stage” MAGE-6 epitopeswhere disease-state, but not disease-stage correlations were noted.

Th3/Tr CD4⁺ T cell subsets may play dominant roles as antigen-specific T“suppressor” cells, in part due to secretion of immunosuppressivecytokines such as TGF-β and/or IL-10 (Krause I, et al. Crit. Rev Immunol20; 1-16, 2000). Based on detection of TGF-β (but not IL-10) productionin 3 of 8 (38%) patients with Stage IV disease, it is possible that thepopulation of human CD4⁺CD25⁺ T suppressor cells may hinder thepatient's ability to productively eliminate EphA2-overexpressing tumors(Levings M K, et al. J Exp Med. 196; 1335-1346, 2002). These samepatients failed to exhibit discernable Th1-type or Th2-type reactivityto EphA2 peptides, supporting the overall suppressive dominance ofEphA2-specific T suppressor-type immunity over Th1- or Th2-typeresponses. These results suggest that Th2- or T suppressor-typeresponses are prevalent against EphA2 epitopes in advanced Stage RCCpatients and likely contribute to the hyporeactivity of tumor-specificcellular immunity noted in these individuals. Future studies could testthis hypothesis using flow cytometry analyses to detect HLA-DR4/EphA2peptide tetramer binding and co-expression of CD25, CTLA-4 or theglucocorticoid-induced tumor necrosis factor receptor (as markers of Tsuppressor cells, Levings et al., J Exp Med. 2002).

Immunotherapies

A broad array of therapeutic vaccines are currently active or beingcontemplated for diverse forms of cancer. Constructive immunologicinformation must be gained from all ongoing trials to provide a basisfor an improved design. Hence, there is a great need for the developmentof innovative methods for the immunological monitoring ofclinically-important T cell responses, which could ultimately serve as“surrogate” endpoints. While no single assay is likely to provesufficiently comprehensive, it is shown herein that the combination ofIFN-γ and IL-5 ELISPOT assays and TGF-β ELISAs provides a sensitivemeans of evaluating functional T cell responses from patients with RCCor melanoma (Tatsumi et al., J. Exp. Med. 2002). These assays areamenable to in vitro detection and frequency determination of both CD8⁺and CD4⁺ T cells specific for tumor-associated antigens. Using suchtechniques, our novel EphA2-derived T cell epitopes may prove useful inevaluating tumor-specific immunity in the many different cancer types inwhich EphA2 overexpression has been documented.

These same epitopes clearly also have potential to serve as componentsof a cancer vaccine. Unlike MAGE-6 reactive T cells, which are skewedtoward Th2-type responses in early-stage disease (Tatsumi et al., J.Exp. Med. 2002), the imbalance in Th reactivity associated with EphA2does not appear to occur until later-stage disease. Hence, EphA2-basedadjuvant vaccination of Stage I patients could have utility foreliciting protective immunity in patients at high risk for diseaserecurrence or to prevent prospective metastases. Vaccination with bothEphA2-derived CD4⁺ and CD8⁺ T cell epitopes may prompt high frequencyanti-EphA2 CTL induction that is stabilized by the concurrent activationof specific Th1-type CD4⁺ T cells. Alternatively under appropriatere-polarizing or activating conditions (Vieira P L, et al. J Immunol164; 4507-4512, 2000), dendritic cell (DC)-based vaccines incorporatingEphA2 peptides may allow for previously muted Th1-type immunity to befunctionally resurrected in patients with advanced stage disease,yielding potential therapeutic benefit.

Given its broad range of EphA2 overexpression among advanced-stagetumors of diverse histologies, vaccines based on antigens such as EphA2have tremendous potential in high-incidence tumor types such as breast,prostate, colon and lung cancer, and in extremely aggressive cancers,such as pancreatic carcinoma (where we have recently observed a 100%incidence of EphA2 overexpression, data not shown). Autologous DC-EphA2vaccines are currently under development for the treatment of patientswith RCC, melanoma, prostate, head and neck or pancreatic cancer.

Example 2 Conditional Triggering of Specific CD8+ T-Cell Recognition ofEphA2 Tumors In Vitro and In Vivo after Treatment with Ligand Agonists

Cell Lines and Media. The T2.DR4 (HLA-A2+/−DRB1*0401+ cell line was usedas the peptide-presenting cell in ELISPOT assays. The EphA2+ HLA-A2-PC-3prostate carcinoma cell line was used as positive control for WesternBlot analysis of EphA2 protein expression and was also used as anegative control target in ELISPOT assays. SLR24, an EphA2+ HLA-A2+ cellline (Tatsumi, T., et al. Cancer Res, 63: 4481-4489, 2003) was tested inWestern Blot and ELISPOT assays and was also applied in the Hu-SCIDtreatment model. All cell lines were maintained in RPMI-1640 culturemedium supplemented with 10% heat-inactivated fetal bovine serum (FBS),100 U/ml penicillin, 100 μg/ml streptomycin and 10 mM L-glutamine (allreagents from GIBCO/Life Technologies, Grand Island, N.Y.) in ahumidified atmosphere under 5% CO₂ tension at 37° C.

Mice. Six-to-eight week old female C.B-17 scid/scid mice were purchasedfrom Taconic Labs (Germantown, N.Y.), and maintained in micro-isolatorcages. Animals were handled under aseptic conditions per anInstitutional Animal Care and Use Committee (IACUC)-approved protocoland in accordance with recommendations for the proper care and use oflaboratory animals.

Western Blot Analyses. Tumor cells were grown to 80-90% confluency,serum starved overnight, then treated with agonists where indicated. Inaddition, resected SLR24 lesions were obtained pre- and 24 hpost-intratumoral injection with B61-Ig, as outlined below. Tumorsamples were analyzed for EphA2 expression via Western blots using therabbit anti-human EphA2 polyclonal antibody (clone: C-20), Santa CruzBiotechnology, Inc., Santa Cruz, Calif.). In some experiments, sampleswere also analyzed for Axl (clone C-20, Santa Cruz Biotechnology, SantaCruz, Calif.) protein content. Single tumor cell suspensions isolatedfrom confluent tissue culture flasks or from the enzymatic digestion ofresected lesions were lysed using 500 μl lysis buffer (1% Triton-X, 150nM NaCl, 10 mM Tris pH7.4, 1 mM EDTA, 0.2 mM SOV, 0.5% NP-40) in PBScontaining protease inhibitors (Complete, Roche Diagnostic, Mannheim,Germany) for 30 min at 4° C. After centrifugation at 13,500×g for 20minutes, the supernatant was mixed 1:1 with SDS-PAGE running buffer andproteins separated on 7.5% PAGE gels, prior to electro-blotting ontonitrocellulose membranes (Millipore, Bedford, Mass.). Blots were imagedon Kodak X-Omat Blue XB-1 film (NEN Life Science Products, Boston,Mass.) after using horseradish peroxidase (HRP)-conjugated goatanti-rabbit Ig (Biorad, Hercules, Calif.) and the Western Lighting™.chemiluminescence detection kit (Perkin Elmer, Boston, Mass.).Immunoprecipitation for EphA2 were performed using the anti-EphA2antibody D7 (Upstate Biotech, Inc.). Anti-phosphotyrosine antibodies(Clone py99, Santa Cruz Biotechnology, San Diego, Calif.) were used toassess pEphA2 content. Mouse anti-β-actin antibody (clone AC-15, Abcam,Cambridge, Mass.) was used as a loading control.

EphA2 Agonists. B61.Ig and mAb208 were kindly provided by MedImmune(Gaithersburg, Md.), B61.Ig is a chimeric protein consisting of theligand binding domain of ephrin-A1 fused with the Fc portion of a mouseIgG antibody and was used in in vitro assays at 30 μg/ml, whereindicated. mAb208 is a mouse monoclonal antibody specific for EphA2 andwas used in in vitro assays at 8 μg/ml, where indicated.

Anti-EphA2 CD8+ T Cell Clones. The CL-142 and E883, HLA-A2-restrictedCD8+ human T cell clones specific for EphA2₈₈₃₋₈₉₁, were generated aspreviously described (Tatsumi, T., et al. Cancer Res. 2003).

ELISPOT Assays. In vitro T cell responses were evaluated by IFN-γELISPOT assays as previously described (Tatsumi, T., et al. Cancer Res.2003). Briefly, 96-well multiscreen hemagglutinin antigen plates(Millipore, Bedford, Mass.) were coated with 10 μg/ml of anti-humanIFN-γ mAb (1-D1K; Mabtech, Stockholm, Sweden) in PBS (GIBCO/LifeTechnologies) overnight at 4° C. Unbound antibody was removed by foursuccessive washing with PBS. After blocking the plates withRPMI-1640/10% human serum (1 hr at 37° C.), 10⁵ CD8+ T cells and T2.DR4cells (2×10⁴ cells) pulsed with 10 μg/ml EphA2₈₈₃₋₈₉₁ peptide(TLADFDPRV, SEQ ID NO: 2, residues 883-891) or SLR24+/−treatmentovernight with B61.Ig were seeded in triplicate in multi-screenhemagglutinin antigen plates. Control wells contained CD8+ with T2.DR4cells pulsed with HIV-nef₁₉₀₋₁₉₈ peptide (AFHHVAREL, SEQ ID NO: 3) orPC3, an HLA-A2-EphA2+ tumor cell line, or T2.DR4 cells alone. Culturemedium (AIM-V; GIBCO/Life Technologies) was added to yield a finalvolume of 200 μl/well. The plates were incubated at 37° C. in 5% CO₂ for24 hr for IFN-γ assessments. Cells were removed from the ELISPOT wellsby washing with PBS/0.05% Tween 20 (PBS/T). Captured cytokines weredetected at sites of their secretion by incubation for 2 hr withbiotinylated mAb anti-human IFN-γ (7-B6-1; Mabtec) at 2 μg/ml inPBS/0.5%. Plates were washed six times using PBS/T, andavidin-peroxidase complex (diluted 1:100; Vectastain Elite Kit; VectorLaboratories, Burlingame, Calif.) was added for 1 hr. Unbound complexwas removed by three successive washes using PBS/T, then with threerinses with PBS alone. AEC substrate (3-Amino-9-ethylcarbazol; Sigma,St. Louis, Mo.) was added and incubated for 5 min for the IFN-γ ELISPOT.Spots were imaged using the Zeiss AutoImager.

Flow Cytometry. For phenotypic analysis of control or ligandagonist-treated tumor cells, PE- or FITC-conjugated monoclonalantibodies against HLA class I (W6/32; pan-class I specific; SerotecInc., Raleigh, N.C.) or human CD40 (Ancell Corp., Bayport, Minn.) andappropriate isotype controls (purchased from BD Biosciences, San Jose,Calif.) were used, and flow cytometric analysis was performed using aFACscan (Becton Dickinson, San Jose, Calif.) flow cytometer. The resultsof the flow cytometric analysis are reported in arbitrary meanfluorescence intensity (MFI) units.

Hu-SCID Tumor Model. C.B17-scid/scid mice were injected s.c. in theright flank with 1×10⁶ SLR24 RCC cells and tumors allowed to establishto a size of approximately 30 mm2 (i.e. day 18 post-injection). Thetumor-bearing mice were then randomized into 4 groups (5 animals eachwith comparable tumor sizes) that received either no treatment, a singleintratumoral injection of 50 μg of B61-Ig (in 50 μl saline) on day 18, asingle tail-vein injection with 5×10⁶ cloned E883 (anti-EphA2₈₈₃₋₈₉₁specific) CD8+ T cells in 100 μl saline on day 19, or the combined d18(B61-Ig) plus d19 (E883 adoptive transfer) regimen. Animals wereevaluated every 3-4 days for tumor size, with tumor-free status noted onday 40 post-tumor inoculation. For the analyses of EphA2 content inSLR24 tumor lesions pre- and post-administration of B61-Ig, tumors weresurgically resected from euthanized mice, digested into single-cellsuspensions using a DNAse, hyaluronidase, DNAse cocktail as previouslydescribed (Itoh, T., et al. J. Immunol. 153: 1202-1215, 1994) andfiltered through Nitex mesh (Tetko, Kansas City, Mo.), prior to tumorcell solublization and Western Blotting, as outlined above.

Statistical Analyses. Statistical differences between groups wereevaluated using a two-tailed Student's T test, with p values <0.05considered significant.

Results

B61.Ig and mAb208 Induce EphA2 Phosphorylation and Degradation.

Previous studies have demonstrated that tumor cells have unstablecell-cell contacts and that this impairs the ability of EphA2 tointeract with its ligands on apposing cells. Consequently, the EphA2 inmalignant cells generally is not itself tyrosine phosphorylated.Consistent with this, Western Blot analyses verified that the EphA2 inmalignant cells (e.g., PC3) is weakly phosphorylated. In FIG. 26, PC3(2-4×10⁶) cells were treated at the indicated time points (in min) witheither B61.Ig (30 μg/ml) or mAb208 (8 μg/ml). B61.Ig is a fusion proteinconsisting of the EphA2 binding domain of ephrin-A1 (a major ligand ofEphA2) fused to a human Fc domain. Cellular lysates were resolved bySDS-PAGE and EphA2 protein was immunoprecipitated using the anti-EphA2antibodies D7 in pull-down assays. Western blot analyses were thenperformed using anti-EphA2 and anti-phosphotyrosine antibodies,respectively. Data are representative of 3 independent experimentsperformed. However, treatment of these cells with reagents that can bindEphA2, even in the absence of stable intercellular contacts (agonisticmonoclonal antibodies and artificial ligands), is sufficient to increaseEphA2 phosphotyrosine content. Immunoblotting of cell lysates verifiedthat this treatment subsequently induces EphA2 protein degradation. Toverify equal loading, the membranes were probed with antibodies specificfor β-actin, which did not change in response to EphA2 agonisttreatment. The specificity for EphA2 was further verified by showingthat the levels of the Axl receptor tyrosine kinase were not altered inresponse to EphA2-specific reagents. In FIG. 27, PC3 (left panel) andSLR24 (right panel) cells were treated for 6 hours with either B61.Ig(30 μg/ml) or mAb208 (8 μg/ml) at 37° C. Cell lysates were resolved by12.5% SDS-PAGE and Western blot analyses were performed using polyclonalanti-EphA2 and control anti-β-actin antibodies. Anti-AXL antibodies wereused to image identically-prepared lysates as a specificity control inthese experiments. Data are representative of 3 independent experimentsperformed on each tumor cell line. Comparable findings were obtained inmultiple and different EphA2-overexpressing cell systems, including cellmodels of breast, lung, pancreatic and renal cell carcinoma (FIG. 27 anddata not shown).

Based on evidence that ligand-mediated stimulation of EphA2 inducesreceptor internalization and degradation within proteasomes, thesefindings were verified and extended to show that antibody stimulationsimilarly induces proteasomal cleavage of EphA2. In FIGS. 28A and 28BPC3 cells were either not treated or treated with B61.Ig (FIG. 28A) ormAb208 (FIG. 28B), as described previously with respect to FIG. 26.MG-132 (50 μM) and chloroquine (Chl.; 100 μM) were also added tocultures, where indicated, 30 min. prior to the addition of EphA2agonists and remained in the cultures for the duration of the 24 hexperiment. Cell lysates were generated and resolved using SDS-PAGE.Western blot analyses were then performed using anti-EphA2 antibodiesand negative control anti-β-actin antibodies. Data are representative of3 independent experiments performed. EphA2 degradation was blocked bythe treatment with the 26S proteasome inhibitor, MG-132. In contrast,the addition of the endosomal/lysosomal inhibitor chloroquine did notprevent EphA2 degradation, thus indicating that proteasomal and notlysosomal degradation of EphA2 is the major mechanism responsible forEphA2 degradation.

EphA2 Agonist Treatment Enhances CD8⁺ T Cell Recognition of EphA2⁺Tumors in Vitro. Since agonistic antibodies triggered proteasomaldegradation of EphA2, it was hypothesized that this could increasepresentation of EphA2 peptides on HLA class I complexes. If correct,then it logically would follow that agonism of EphA2 could enhancerecognition by EphA2-specific CD8⁺ T cells. To address this question,EphA2⁺ SLR24 RCC cells were incubated with B61-Ig prior to evaluatingtheir ability to be recognized by the EphA2₈₈₃₋₈₉₁ specific CTL clone142 (CL.142). As a readout of T cell activation, the samples weresubjected to IFN-γ-based ELISPOT assays. In FIG. 29, the anti-EphA2 CTLclone CL142 (Dobrzanski, P., et al. Cancer Res. 64: 910-919, 2004) wasanalyzed for reactivity against T2.DR4 (A2⁺) cells pulsed with theEphA2₈₈₃₋₈₉₁ peptide epitope, or against untreated or agonist-triggeredHLA-A2⁺/EphA2⁺ SLR24 cells as targets in IFN-γ ELISPOT assays. Controltarget cells include: T2.DR4 cells pulsed with the HLA-A2-presentedHIV-nef₁₉₀₋₁₉₈ (negative control for peptide specificity) and PC3(HLA-A2⁻/EphA2⁺) prostate carcinoma cells. B61.Ig treatment (30 μg/ml)was applied overnight to ensure EphA2 degradation and HLA antigenprocessing and presentation of EphA2 epitopes). Data are reported atIFN-γ specific spots/10,000 CL.142 cells and are derived from onerepresentative experiment of 3 performed.

Pretreatment of SLR24 with B61.Ig significantly enhanced CL.142recognition of SLR24 relative to untreated control cells. It was thenconsidered that the increased tumor cell recognition could have been dueto changes in tumor cell expression of HLA class I or costimulatorymolecules. To address this, SLR24 cells were treated with agonisticantibodies and surface levels of HLA class I and CD40 evaluated by flowcytometry. Notably, the staining intensity of both HLA class I and CD40was not significantly altered following agonism of EphA2 (Table 4). As afurther control for the selectivity of this effect, it was observed thatrecognition of SLR24 tumor cells by HLA-A2 alloreactive CTLs was notaltered pre- vs. post-treatment with EphA2 agonists (data not shown).

TABLE 4 EphA2 Agonists Affect HLA Class I or CD40 Expression on SLR24tumor cells Mean Fluorescence Intensity Treatment MG-132 (+/−) ControlW6/32 CD40 Untreated − 0.5 124.7 14.8 MAb208 − 5.5 116.5 19.8

The SLR24 RCC cell line was either not treated or treated with mAb208(10 μg/ml) as outlined in the description of FIGS. 28A and 28B, above.Treated cells were then analyzed for expression of HLA class I and CD40molecules by flow cytometry as described in Materials and Methods. Datapresented is the mean fluorescence intensity of expression for theindicated markers.

EphA2 Agonist Treatment Enhances the efficacy of adoptively transferredanti-EphA2 CD8⁺ T Cells in a Hu-SCID Tumor Model. To determine whetherthe conditional (agonist-induced) increase in anti-EphA2 CD8⁺ T cellreactivity against EphA2⁺ tumors could be of potential clinicalsignificance, a Hu-SCID tumor model system was established. SLR24 tumorswere injected into C.B-17 scid/scid mice and allowed to progress to asize of approximately 30 mm², at which time, animals were either leftuntreated, or treated with intratumoral injection of B61-Ig (day 18post-tumor inoculation) and/or intravenous delivery of aHLA-A2-restricted, anti-EphA2883-891 CD8+ T cell clone (E883). In FIG.30A; Female CB17-scid/scid mice were injected with 1×10⁶ human SLR24(HLA-A2+/EphA2+) RCC cells s.c. in the right flank and allowed toestablish to a size of approximately 30 mm² (i.e. dl 8). Animals werethen randomized into 4 cohorts (5 animals each) receiving no treatment(control), intratumoral injection of B61-Ig (50 μg) on d18 to triggerEphA2 degradation and proteasomal processing, intravenous (tail-vein)injection of 5×10⁶ cloned E883 anti-EphA2883-891 CD8+ T cells on d19, orboth the B61-Ig (d18) and CD8+ T cell (d19) injections. Animals wereevaluated every 3-4 days for tumor size, with tumor-free status noted onday 40 post-tumor inoculation. Arrows indicate treatment days. In FIG.30B, in repeat experiments, tumors were resected from animals on day 19(i.e. 24 hours after B61-Ig administration and Western Blots performedto validate EphA2 degradation in situ.

As depicted in FIG. 30A, while the administration of either B61-Ig orE883 T cells promoted the delayed growth of SLR24 tumors, no animalswere cured by these therapies. In marked contrast, the combinationaltherapy including B61-Ig delivery (which promoted EphA2 processing insitu, FIG. 30B) and the adoptive transfer of anti-EphA2 CD8+ T cells,promoted the rapid resolution of disease in 5/5 treated mice. In controlcohorts, B61-Ig treatment combined with the adoptive transfer of clonedCD8+ T cells specific for the HLA-A2-presented influenza matrix₅₈₋₆₆epitope, provided no enhanced benefit vs. B61-Ig treatment alone (datanot shown).

The major finding of the present study is that the treatment of tumorcells with agonists that promote EphA2 autophosphorylation andproteasomal processing also result in improved recognition byEphA2-specific CD8+ T-cells both in vitro and in vivo. As a consequence,EphA2-reactive CD8+ T cells are rendered more effective in mediating theregression of tumor lesions in situ.

In normal epithelia, EphA2 localizes to cell-cell boundaries, where itconstitutively binds its ligands. Consequently, the EphA2 innon-transformed cells is tyrosine phosphorylated and mediates signalsthat serve to limit epithelial cell growth. In particular,phosphorylated EphA2 molecules form signaling complexes with adapterproteins that contain SH2 domains (e.g., c-Cbl, SHC, SLAP, and GRB2)alters enzymatic activity of selected downstream effectors (e.g., FAK,SHP-2, PI 3-kinase, LMW-PTP). These signals, in turn, decrease theability of EphA2⁺ epithelial cells to establish or maintain stablecontacts with the surrounding extracellular matrix (ECM).

The interaction with c-Cbl is particularly relevant to the presentfindings. c-Cbl contains an ubiquitin-E3 ligase and targets proteins fordegradation via the proteasome. The results herein indicate thatproteasomal degradation increases T cell recognition of EphA2,presumably by cleaving EphA2 into peptides that are loaded into HLAcomplexes for subsequent antigen presentation to effector T cells.

Tumor cells generally have unstable cell-cell contacts, which appears topreclude access of EphA2 to its membrane-anchored ligands. This isconsistent with experimental evidence that the EphA2 in tumor cells isoverexpressed, but unphosphorylated. Compounding decreased ligandbinding, EphA2 molecules expressed by tumor cells serve as substratesfor certain oncogenic tyrosine phosphatases, which provide an additionalmeans of decreasing EphA2 phosphotyrosine content. Regardless of thecause, decreased phosphotyrosine content causes the EphA2 in tumor cellsto increase their malignant character. In part, the increasedinvasiveness relates to increased tumor cell interactions with the ECM.These changes are frequently observed in clinical specimens of cancer.Under such conditions, EphA2 ligand agonists can restore a normalizedpattern of contact inhibited growth and reduce the invasiveness ofEphA2+ tumor cells.

The ability of agonistic reagents to conditionally and specificallytrigger EphA2 degradation provide opportunities for the development ofnew therapeutic strategies for the treatment patients with EphA2⁺cancers. In particular, these results suggest that clinical impact ofendogenous, anti-EphA2 T cell-mediate immunity could be enhanced bycombining passive and active EphA2-specific immunotherapies. Previousstudies have shown that a subset of T cells isolated from cancerpatients can be stimulated following presentation of EphA2 peptides onHLA molecules (Tatsumi, T., Cancer Res. 2003 and Alves, P. M. et al.Cancer Res. 63:8476-8480, 2003). These studies were conducted using exvivo stimulation of T cells using DC-based vaccination approaches, whichcould certainly be employed in the clinic.

Whereas, particular embodiments of the invention have been describedabove for purposes of description, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

We claim:
 1. A method of treating cancer in a patient in need thereof,said method comprising administering to the patient a therapeuticallyeffective amount of a purified EphA2 T-cell epitope, wherein said EphA2T-cell epitope binds to an MHC Class II molecule, wherein said EphA2T-cell epitope is a peptide that consists of 9 to 35 contiguous aminoacid residues of native human EphA2 (SEQ ID NO: 2), and wherein saidEphA2 T-cell epitope has a predicted binding score of greater than 39for the MHC Class II molecule HLA-DRβ1*0402 as determined using theSouthwood algorithm.
 2. A method of treating cancer, said methodcomprising administering to a patient in need thereof a therapeuticallyeffective amount of a purified EphA2 T-cell epitope or a purifiedpolypeptide comprising an EphA2 T-cell epitope and a second amino acidsequence, wherein the EphA2 T-cell epitope is a peptide that: (i)consists of 9 to 35 contiguous amino acid residues of native human EphA2(SEQ ID NO: 2), and (ii) has: (x) a predicted binding score of 98 orbelow for the major histocompatibility complex (“MHC”) Class I moleculeHLA-A2 as determined using the NIH BIMAS “HLA Peptide BindingPredictions” software, or (y) an amino acid sequence identified usingthe ProPred software as a sequence that binds to MHC Class II moleculeHLA-DRβ1*0402, wherein said EphA2 T-cell epitope and said second aminoacid sequence are not contiguous to each other in the amino acidsequence of SEQ ID NO:
 2. 3. A method of eliciting an immune response toEphA2 in a patient, said method comprising administering to the patientan effective amount of a purified EphA2 T-cell epitope or a purifiedpolypeptide comprising an EphA2 T-cell epitope and a second amino acidsequence, wherein the EphA2 T-cell epitope is a peptide that: (i)consists of 9 to 35 contiguous amino acid residues of native human EphA2(SEQ ID NO: 2), and (ii) has: (x) a predicted binding score of 98 orbelow for the major histocompatibility complex (“MHC”) Class I moleculeHLA-A2 as determined using the NIH BIMAS “HLA Peptide BindingPredictions” software, or (y) an amino acid sequence identified usingthe ProPred software as a sequence that binds to MHC Class II moleculeHLA-DRβ1*0402, wherein said EphA2 T-cell epitope and said second aminoacid sequence are not contiguous to each other in the amino acidsequence of SEQ ID NO:
 2. 4. The method of claim 2 or 3, wherein thesecond amino acid sequence is a heterologous amino acid sequence.
 5. Themethod of claim 2 or 3, wherein the second amino acid sequence is asecond EphA2 T-cell epitope.
 6. The method of claim 5, wherein saidsecond EphA2 T-cell epitope: (i) binds to an MHC Class II molecule, (ii)consists of 9 to 35 contiguous amino acid residues of native human EphA2(SEQ ID NO:2), and (iii) has: (x) a predicted binding score of greaterthan 39 for the MHC Class II molecule HLA-DRβ1*0401 as determined usingthe Southwood algorithm, or (y) an amino acid sequence identified usingthe ProPred software as a sequence that binds to MHC Class II moleculeHLA-DRβ1*0402.
 7. The method of claim 5, wherein said second EphA2T-cell epitope binds to an MHC Class II molecule and has an amino acidsequence selected from the group consisting of: WLVPIGQCL (SEQ ID NO: 2,residues 253-261), LLWGCALAA (SEQ ID NO: 2, residues 12-20), NLYYAESDL(SEQ ID NO: 2, residues 120-128), MQNIMNDMP (SEQ ID NO: 2, residues55-63), DLMQNIMNDMPIYMYS (SEQ ID NO: 2, residues 53-68), IMGQFSHHN (SEQID NO: 2, residues 666-674), IVMWEVMTY (SEQ ID NO: 2, residues 805-813),IVYSVTCEQ (SEQ ID NO: 2, residues 360-368), IRLPSTSGS (SEQ ID NO: 2,residues 893-901), VELRWTPPQ (SEQ ID NO: 2, residues 344-352), LRWTPPQDS(SEQ ID NO: 2, residues 346-354), VVLLLVLAG (SEQ ID NO: 2, residues545-553), ILVNSNLVC (SEQ ID NO: 2, residues 745-753), and MNYTFTVEA (SEQID NO: 2, residues 406-414).
 8. The method of claim 5, wherein thesecond EphA2 T-cell epitope has an amino acid sequence selected from thegroup consisting of: IMGQFSHHN (SEQ ID NO: 2, residues 666-674);YSVCNVMSG (SEQ ID NO: 2, residues 67-75); EAGIMGQFSHHNIIR (SEQ ID NO: 2,residues 663-677); and PIYMYSVCNVMSG (SEQ ID NO: 2, residues 63-75). 9.A method of treating cancer in a patient in need thereof, said methodcomprising administering to the patient a therapeutically effectiveamount of a purified EphA2 T-cell epitope, wherein said EphA2 T-cellepitope is a peptide that consists of 9 to 35 contiguous amino acidresidues of native human EphA2 (SEQ ID NO: 2), and is selected from thegroup consisting of: TLADFDPRV (SEQ ID NO: 2, residues 883-891);VLLLVLAGV (SEQ ID NO: 2, residues 546-554); VLAGVGFFI (SEQ ID NO: 2,residues 550-558); IMNDMPIYM (SEQ ID NO: 2, residues 58-66); SLLGLKDQV(SEQ ID NO: 2, residues 961-969); WLVPIGQCL (SEQ ID NO: 2, residues253-261); LLWGCALAA (SEQ ID NO: 2, residues 12-20); GLTRTSVTV (SEQ IDNO: 2, residues 391-399); NLYYAESDL (SEQ ID NO: 2, residues 120-128);KLNVEERSV (SEQ ID NO: 2, residues 162-170); IMGQFSHHN (SEQ ID NO: 2,residues 666-674); YSVCNVMSG (SEQ ID NO: 2, residues 67-75); MQNIMNDMP(SEQ ID NO: 2, residues 55-63); EAGIMGQFSHHNIIR (SEQ ID NO: 2, residues663-677); PIYMYSVCNVMSG (SEQ ID NO: 2, residues 63-75); DLMQNIMNDMPIYMYS(SEQ ID NO: 2, residues 53-68); DLMQNIMNDMPIYMYSVCNVMSG (SEQ ID NO: 2,residues 53-75); and VLLLVLAGVGFFI (SEQ ID NO: 2, residues 546-558).